JP6793699B2 - CRISPR enzyme mutations that reduce off-target effects - Google Patents

Description

Mutual Reference / Incorporation by Reference US Provisional Patent Application No. 62 / 181,453 filed June 18, 2015, US Provisional Patent Application No. 62 / 207,312 filed August 19, 2015. Specification, US Provisional Patent Application No. 62 / 237,360 filed on October 5, 2015, US Provisional Patent Application No. 62 / 255,256 filed on November 13, 2015. See also US Provisional Patent Application No. 62 / 269,876, filed December 18, 2015.

The above applications, and all documents cited in these applications or cited during examination of these applications (“Application Cited References”), and all documents cited or referenced in this application cited document, and All references cited or referred to herein (“references cited herein”), and all references cited or referred to in the references cited herein, are referred to herein or herein. Incorporated herein by reference and in practice of the present invention, along with any manufacturer's instructions, instructions, product specifications, and product sheets for any product described in any document incorporated by reference. It can be used. More specifically, all references are incorporated by reference to the same extent that each individual document is shown to be specifically and individually incorporated by reference.

Matters Concerning Federal-Supported Research The present invention was made with the support of the federal government under grants MH100706 and MH110049 granted by the National Institutes of Health. The federal government has certain rights to the invention.

The present invention generally relates to clustered, regularly spaced short palindrome repeats (CRISPR), CRISPR enzymes (eg, Cas or Cas9), CRISPR-Cas or CRISPR systems or CRISPR-Cas composites, among other aspects in general. With respect to the body, its constituents, the nucleic acid molecules involved, such as vectors, and all of the aforementioned uses.

The first publication of a feasible disclosure of how to make and use the CRISPR-Cas system in eukaryotic cells was published in Kong et al. , Science 2013; 339: 819-823 (online publication 3 January 2013). The first patent application for feasible disclosure of how to make and use the CRISPR-Cas system in eukaryotic cells was filed on December 12, 2012 by Zhang et al. , US Provisional Patent Application No. 61 / 736,527, many of which are original US Patent Nos. 8,999,641 and 8,993,233. , 8,945,839, 8,923,814, 8,906,616, 8,895,308, 8,889. , No. 418, No. 8,889,356, No. 8,871,445, No. 8,856,406, No. 8,795,965, It claims priority in the future, including those matured in the same No. 8,771,945 and No. 8,697,359.

In line with the breakthroughs that enable the use of the CRISPR-Cas system in eukaryotic cells, Broad Institute's Zhang et al. The laboratory was used to cause alterations in target loci, but there was recognition that an improved CRISPR enzyme was still needed that reduced or eliminated activity against off-targets. There is an urgent need for alternative and robust systems and techniques that reduce the off-target activity of CRISPR enzymes when forming complexes with guide RNAs. There is also an urgent need for alternative and robust systems and techniques that increase the activity of CRISPR enzymes when complexed with guide RNAs.

Decreasing the amount of Cas9 in cells to enhance Cas9 specificity, creating adjacent single-stranded DNA nick pairs using Cas9 nickase mutants, truncating guide sequences at the 5'end Several strategies have been developed, including using a pair of catalytically inactive Cas9 nucleases, each fused to the FokI nuclease domain.

Surprisingly, we have determined that a modification can be made to a CRISPR enzyme that imparts a decrease in off-target activity compared to an unmodified CRISPR enzyme and / or an increase in target activity compared to an unmodified CRISPR enzyme. .. Accordingly, the present specification provides an improved CRISPR enzyme that may be useful for a wide range of genetically modified applications. Also provided herein are CRISPR complexes, compositions and systems, as well as methods and uses, all of which include the modified CRISPR enzymes disclosed herein. Without limitation, CRISPR-Cas9 is preferred, including SaCas9, SpCas9, and orthologs.

In some embodiments, an engineered CRISPR protein is provided, where the protein is complexed with a nucleic acid molecule containing RNA to form a CRISPR complex, where the nucleic acid molecule is 1 when it is within the CRISPR complex. Targeting one or more target polynucleotide loci, the protein comprises at least one modification as compared to the unmodified CRISPR, and the CRISPR complex comprising the modified protein is here with the complex comprising the unmodified CRISPR protein. The activity has changed when compared. Without limitation, CRISPR-Cas9 is preferred, including SaCas9, SpCas9, and orthologs. CRISPR proteins include those with enzymatic activity, eg, nuclease activity.

In some embodiments, changes in the activity of engineered CRISPR proteins include changes in binding properties for RNA-containing nucleic acid molecules or target polynucleotide loci, RNA-containing nucleic acid molecules, or RNA-containing nucleic acid molecules as compared to off-target polynucleotide loci. Included are changes in binding reaction rate for the target polynucleotide locus, or changes in binding specificity for the nucleic acid molecule containing RNA or the target polynucleotide locus.

In certain embodiments, changes in the activity of the engineered CRISPR protein include increased targeting efficiency or decreased off-target binding. In certain embodiments, changes in the activity of engineered CRISPR proteins include alterations in cleavage activity.

In certain embodiments, changes in activity include increased cleavage activity for the target polynucleotide locus. In certain embodiments, changes in activity include reduced cleavage activity for the target polynucleotide locus. In certain embodiments, changes in activity include reduced cleavage activity for off-target polynucleotide loci. In certain embodiments, changes in activity include increased cleavage activity for off-target polynucleotide loci. Therefore, in certain embodiments, the specificity for the polynuucleotide locus is increased when compared to the off-target polynucleotide locus. In other embodiments, the specificity for the polyneucleotide locus is reduced when compared to the off-target polynucleotide locus.

In certain aspects of the invention, changes in the activity of the engineered CRISPR protein include changes in helicase kinetics.

In certain aspects of the invention, the engineered CRISPR protein comprises a modification that alters the association of the protein with a nucleic acid molecule, including RNA, or a target polynucleotide locus, or an off-target polynucleotide locus. In certain aspects of the invention, the engineered CRISPR protein comprises modifications that alter the formation of the CRISPR complex.

The present invention
Provides a non-naturally occurring CRISPR enzyme, where:
This enzyme complexes with the guide RNA to form the CRISPR complex,
When in the CRISPR complex, the guide RNA targets one or more target polynucleotide loci, the enzyme alters the polynucleotide locus, and the enzyme comprises at least one modification.
This reduces the ability of the enzyme in the CRISPR complex to modify one or more off-target loci when compared to the unmodified enzyme, and / or thereby the enzyme in the CRISPR complex when compared to the unmodified enzyme 1 Increases the ability to modify one or more target loci.

In any such non-naturally occurring CRISPR enzyme, modifications may include modifications of one or more amino acid residues of the enzyme.

In any such non-naturally occurring CRISPR enzyme, the modification may include modification of one or more amino acid residues located in the region containing the positively charged residue in the non-modifying enzyme.

In any such non-naturally occurring CRISPR enzyme, the modification may include modification of one or more positively charged amino acid residues in the non-modifying enzyme.

In any such non-naturally occurring CRISPR enzyme, modifications may include modifications of one or more amino acid residues that are not positively charged in the unmodified enzyme.

Modifications can include modification of one or more uncharged amino acid residues in an unmodified enzyme.

Modifications can include modification of one or more negatively charged amino acid residues in a non-modifying enzyme.

Modifications can include modification of one or more amino acid residues that are hydrophobic in a non-modifying enzyme.

Modifications can include modification of one or more polar amino acid residues in a non-modifying enzyme.

In any of the non-naturally occurring CRISPR enzymes described above, the enzyme can include type II CRISPR enzymes. The enzyme may include Cas9 enzyme.

In certain of the non-naturally occurring CRISPR enzymes described above, modifications may include modifications of one or more residues located in the region between the RuvC and HNH domains. The RuvC domain may include a RuvCII domain or a RuvCIII domain. Modifications can include modifications of one or more residues located in the groove.

In certain of the non-naturally occurring CRISPR enzymes described above, modifications may include one or more residue modifications located outside the region between the RuvC and HNH domains, or outside the groove.

In certain of the above-mentioned non-naturally occurring CRISPR enzymes, modifications include regions:
Streptococcus pyogenes Cas9 (SpCas9) residues R63-K1325 or K775-K1325 or corresponding regions in another Cas9 ortholog; or Staphylococcus aureus Cas9 (SaCas9) Cas9 (SaCas9) Corresponding area in another Cas9 ortholog,
Modifications of one or more residues in.

In certain non-naturally occurring CRISPR enzymes described above, modifications include modifications of one or more residues, the one or more residues including arginine, histidine or lysine.

In any of the non-naturally occurring CRISPR enzymes described above, the enzyme can be modified by mutation of one or more of the residues.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where this mutation replaces the residue in the unmodified enzyme with an alanine residue. including.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where the mutation is of a residue in the unmodified enzyme with aspartic acid or glutamic acid. Includes substitution.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the residues, where the mutation is in a non-modifying enzyme with serine, threonine, asparagine or glutamine. Includes residue substitution.

In certain non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the residues, where the mutations are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, tryptophan. Includes substitution of residues in unmodified enzymes with, tyrosine or valine.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where this mutation is a residue of a non-modifying enzyme with polar amino acid residues. Includes substitution.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where the mutation is in an unmodified enzyme with an amino acid residue that is not a polar amino acid residue. Includes substitution of residues in.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where this mutation is a residue in the unmodified enzyme with a negatively charged amino acid residue. Includes replacement of.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the residues, where the mutation is an unmodified enzyme with amino acid residues that are not negatively charged amino acid residues. Includes substitution of residues in.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where this mutation is a residue in the unmodified enzyme with uncharged amino acid residues. Includes replacement of.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the residues, where the mutation is an unmodified enzyme with an amino acid residue that is not an uncharged amino acid residue. Includes substitution of residues in.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where this mutation is a residue in the unmodified enzyme with hydrophobic amino acid residues. Includes replacement of.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more of the above residues, where this mutation is an unmodified enzyme with amino acid residues that are not hydrophobic amino acid residues. Includes substitution of residues in.

The non-naturally occurring CRISPR enzyme may be a SpCas9 or SpCas9 ortholog, and here:
This enzyme is modified or contains modifications, eg, by mutation of any one of the SpCas9 or SaCas9 residues listed in any one of Tables 1-7 or the corresponding residue in the Cas9 ortholog. Containing, or essentially consisting of, modifications; or, without limitation, this enzyme is a brief description of this overview and / or drawing and / or of embodiments and / or embodiments for carrying out the invention. Any one (single), two (double), three (triple), four (quadruple) or more in this disclosure throughout the present application, including any and / or any of the figures. Containing, or essentially becoming, or consisting of modifications to the position of, or the corresponding residue or position in the Cas9 ortholog, eg, any brief description and / or invention of the present invention and / or drawings. Any one of the embodiments and / or examples to be performed and / or any one of the Cas9 residues described elsewhere in the figure, or the corresponding residue in the Cas9 ortholog. An enzyme that contains or consists of alterations in position. In such enzymes, each residue may be modified by substitution with an alanine residue.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is, but is not limited to, positions 12, 13, 63, 415, 610, 775, 779, 780, 810, relative to the amino acid position numbering of SpCas9. 832, 848, 855, 861, 862, 866, 961, 968, 974, 976, 982, 983, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, It is modified by mutation of one or more residues, including 1297, 1300, 1311, and 1325.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation and, but is not limited to, positions 63, 415, 775, 779, 780, 810, 832 relative to the amino acid position numbering of SpCas9. , 848, 855, 861, 862, 866, 961, 968, 974, 976, 982, 983, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, 1297. Residues containing 1,300, 1311, or 1325 contain one or more alanine substitutions.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is mutated and modified by mutations such as K775A, E779L, Q807A, R780A, K810A, R832A, K848A, K855A, K862A, K866A, K961A, K968A, K974A, R976A, Includes one or more substitutions of H982A, H983A, K1000A, K1014A, K1047A, K1060A, K1003A, K1107A, S1109A, H1240A, K1289A, K1296A, H1297A, K1300A, H1311A, or K1325A.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation and comprises two or more substitutions, the two or more substitutions including, without limitation, R783A and A1322T, or R780A and. K810A, or ER780A and K855A, or R780A and R976A, or K848A and R976A, or K855A and R976A, and R780A and K848A, or K810A and K848A, or K848A and K855A, or K810A and K855A and R98A and K855A, or H. R1003A, or K1003A and R1060A, or R780A and H982A, or K810A and H982A, or K848A and H982A, or K855A and H982A, or R780A and K1003A, or K810A and R1003A, or K848A and K1003A and R1003A, or K848A and K1003A. R1060A, or K810A and R1060A, or K848A and R1060A, or R780A and R1114A, or K848A and R1114A, or R63A and K855A, or R63A and H982A, or H415A and R780A, or H415A and R780A, or H415A and K848A, or K848A. Included are K1003A, or R780A and R1060A, K810A and R1060A, or K848A and R1060A.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation and comprises three or more substitutions, the three or more substitutions including, without limitation, H982A, K1003A, and K1129E. Or R780A, K1003A, and R1060A, or K810A, K1003A, and R1060A, or K848A, K1003A, and R1060A, or K855A, K1003A, and R1060A, or H982A, K1003A, and R1060A, or R63A, K1360A, , R63A, and K810A, or G12D, R63A, and R1060A.

In certain of the non-naturally occurring CRISPR enzymes described above, the enzyme is mutated and comprises four or more substitutions, the four or more substitutions including, without limitation, R63A, E610G, K855A, and. Includes R1060A, or R63A, K855A, R1060A, and E610G.

In a preferred embodiment, mutations in the non-naturally occurring CRISPR enzyme are not the mutations listed in Table B. In a more preferred embodiment, the mutation of the non-naturally occurring CRISPR enzyme is not R63A, K866A, H982A, H983A, K1107A, K1107A, KES1107-1109AG or KES1107-1109GG relative to the amino acid positional numbering of SpCas9. In a more preferred embodiment, the non-naturally occurring CRISPR enzyme is an enzyme modified by a single mutation selected from R63A, K866A, H982A, H983A, K1107A and K1107A based on the amino acid positioning of SpCas9 or KES1107. It is not a mutation-modified enzyme selected from -1109AG and KES1107-1109GG.

In a preferred embodiment, the non-naturally occurring CRISPR enzymes described above are, but are not limited to, positions 12, 13, 415, 610, 775, 779, 780, 810, 832, 848, relative to the amino acid position numbering of SpCas9. One or more residues including 855, 861, 862, 961, 968, 974, 976, 1000, 1003, 1014, 1047, 1060, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311, and 1325. It is modified by the mutation of.

In a more preferred embodiment, the non-naturally occurring CRISPR enzyme described above is mutated and modified with, but not limited to, positions 415, 775, 779, 780, 810, 832, 848, relative to the amino acid position numbering of SpCas9. One or more on residues containing 855, 861, 862, 961, 968, 974, 976, 1000, 1003, 1014, 1047, 1060, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311, or 1325. Includes alanine substitution in.

In a more preferred embodiment, the non-naturally occurring CRISPR enzyme described above is modified by mutation to K775A, E779L, Q807A, R780A, K810A, R832A, K848A, K855A, K862A, K961A, K968A, K974A, R976A, K1000A, K1014A. , K1047A, K1060A, K1003A, S1109A, H1240A, K1289A, K1296A, H1297A, K1300A, H1311A, or K1325A.

In any of the non-naturally occurring CRISPR enzymes:
There may be a single mismatch between the target and the corresponding sequence of one or more off-target loci; and / or between the target and the corresponding sequence of one or more off-target loci. There may be 2, 3 or 4 or more mismatches, and / or where in (ii), the 2, 3 or 4 or more mismatches are continuous.

In any of the non-naturally occurring CRISPR enzymes, the enzyme in the CRISPR complex may have a reduced ability to modify one or more off-target loci when compared to the unmodified enzyme, and the enzyme in the CRISPR complex. Increases the ability to modify the target locus when compared to unmodified enzymes.

In any of the non-naturally occurring CRISPR enzymes, the relative difference in the ability of the enzyme to modify as it is between the target and at least one off-target locus when in the CRISPR complex is that of the unmodified enzyme. Can increase compared to relative differences.

In any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may contain one or more additional mutations, the one or more additional mutations being in one or more catalytically active domains.

In such non-naturally occurring CRISPR enzymes, the CRISPR enzyme may have reduced or abolished nuclease activity as compared to the enzyme lacking the one or more additional mutations.

In some such non-naturally occurring CRISPR enzymes, the CRISPR enzyme does not induce cleavage of either DNA strand at that position in the target sequence.

In some such non-naturally occurring CRISPR enzymes, one or more additional mutations include SpCas9 D10, SpCas9 E762, SpCas9 H840, SpCas9 N854, SpCas9 N863 and / or SpCas9 D986 or other. Includes mutations in the corresponding residues of the Cas9 ortholog.

In some such non-naturally occurring CRISPR enzymes, one or more additional mutations include the corresponding residues of SpCas9 D10A, E762A, H840A, N854A, N863A and / or D986A or other Cas9 orthologs.

In some such non-naturally occurring CRISPR enzymes, one or more additional mutations comprises two additional mutations. The two additional mutations may include D10A SpCas9 and H840A SpCas9, or the corresponding residues of another Cas9 ortholog. In some such non-naturally occurring CRISPR enzymes, the CRISPR enzyme may not lead to cleavage of any DNA strand at that position in the target sequence.

If the CRISPR enzyme contains one or more additional mutations in one or more catalytically active domains, the one or more additional mutations are in the catalytically active domain of the CRISPR enzyme, including RuvCI, RuvCII or RuvCIII. May be good.

Although not constrained by theory, in certain aspects of the invention, the methods and mutations described are conformational reorganizations of Cas9 domains to positions that result in cleavage at the on-target site and their conformation at the off-target site. Provides enhanced avoidance of the condition. Cas9 cleaves the target DNA in a series of coordinated steps. First, the PAM interaction domain recognizes the PAM sequence on the 5'side of the target DNA. After PAM binding, the first 10-12 nucleotides (seed sequence) of the target sequence are sampled for sgRNA: DNA complementarity, a process that relies on the separation of DNA double strands. If the seed sequence nucleotide is complementary to the sgRNA, the remaining DNA is unwound and the full length of the sgRNA hybridizes to the target DNA strand. The nt groove between the RuvC domain and the HNH domain stabilizes the non-target DNA strand by non-specific interaction with the positive charge of the DNA phosphate backbone and promotes unwinding. RNA: cDNA and Cas9: n cDNA interactions drive DNA unwinding in competition with cDNA: nDNA rehybridization. Linkers linking other cas9 domains, such as HNH, to RuvCII and RuvCIII further affect the conformation of the nuclease domain. Thus, the methods and mutations provided include, without limitation, RuvCI, RuvCIII, RuvCIII and HNH domains and linkers. Conformational changes in Cas9 caused by target DNA binding, including seed sequence interactions and interactions with target and non-target DNA strands, determine whether the domain is in a position to elicit nuclease activity. Thus, the mutations and methods provided herein demonstrate and enable alterations that go beyond PAM recognition and RNA-DNA base pairing.

In some embodiments, the invention departs from the improved equilibrium and / or conformation associated with cleavage activity when involved in off-target interactions towards improved equilibrium and / or conformation associated with cleavage activity when involved in on-target interactions. A Cas9 nuclease containing an improved equilibrium is provided. In one aspect, the invention is a Cas9 nuclease with improved calibrating function, i.e., a Cas9 nuclease that takes a conformation containing nuclease activity at the on-target site and increases the disadvantage of that conformation at the off-target site. I will provide a. Sternberg et al. , Nature 527 (7576): 110-3, doi: 10.1038 / nature15544, published online 28 October 2015. EPUB 2015 Oct 28 used Felster resonance energy transfer FRET) experiments to detect the relative orientation of the Cas9 catalytic domain when associated with on-target and off-target DNA.

The present invention further provides methods and mutations that use modified guide RNAs to regulate nuclease activity and / or specificity. As discussed, on-target nuclease activity can be increased or decreased. It can also increase or decrease off-target nuclease activity. In addition, there can be an increase or decrease in specificity with respect to on-target activity vs. off-target activity. The modified guide RNA includes, without limitation, a truncated guide RNA, a dead guide RNA, a chemically modified guide RNA, a guide RNA associated with a functional domain, a modified guide RNA containing a functional domain, and an aptamer. Included are guide RNAs, modified guide RNAs containing adapter proteins, and guide RNAs containing added or modified loops.

In some embodiments, the invention also provides methods and mutations that regulate Cas9 binding activity and / or binding specificity. In certain embodiments, Cas9 proteins lacking nuclease activity are used. In certain embodiments, modified guide RNAs are utilized that promote the binding of Cas9 nuclease but not the nuclease activity. In such an embodiment, on-target binding can be increased or decreased. Also, in such embodiments, off-target binding can be increased or decreased. In addition, there can be an increase or decrease in specificity for on-target binding vs. off-target binding.

Methods and abruptly available in various combinations to increase or decrease the activity and / or specificity of on-target vs. off-target activity, or to increase or decrease the binding and / or specificity of on-target vs. off-target binding. Mutations can be used to compensate or enhance mutations or modifications that are added to promote other effects. Such mutations or modifications that are added to promote other effects include mutations or modifications to Cas9 and / or mutations or modifications that are added to the guide RNA. In certain embodiments, the method and mutations are used with chemically modified guide RNAs. Examples of chemical modifications of guide RNA are, without limitation, 2'-O-methyl (M), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O in one or more terminal nucleotides. -Incorporation of methyl 3'thioPACE (MSP) can be mentioned. Such chemically modified guide RNAs can contain high stability and high activity when compared to unmodified guide RNAs, however, on-target vs. off-target specificity is unpredictable (Hender, 2015, Nat Biotechnology.33 (9): 985-9, doi: 10.1038 / nbt.3290, published online 29 June 2015). Chemically modified guide RNAs further include, without limitation, RNA having a phosphorothioate bond and locked nucleic acid (LNA) nucleotides containing a methylene bridge between the 2'and 4'carbons of the ribose ring. The methods and mutations of the present invention are used to regulate Cas9 nuclease activity and / or binding by chemically modified guide RNAs.

In some embodiments, the invention provides methods and mutations that regulate the binding and / or binding specificity of Cas9 proteins, including functional domains such as nucleases, transcriptional activators, transcriptional repressors. For example, the Cas9 protein can be nuclease null by introducing mutations such as D10A, D839A, H840A and N863A into the nuclease domains RuvC and HNH. The nuclease-deficient Cas9 protein is useful for RNA-guided target sequence-dependent delivery of functional domains. The present invention provides methods and mutations that regulate the binding of Cas9 proteins. In one embodiment, the functional domain comprises VP64, which provides an RNA-guided transcription factor. In another embodiment, the functional domain comprises Fok I, which provides RNA-guided nuclease activity. U.S. Patent Application Publication No. 2014/0356959, U.S. Patent Application Publication No. 2014/03424656, U.S. Patent Application Publication No. 2015/0031132, and Mali, P. et al. et al. , 2013, Science 339 (6121): 823-6, doi: 10.1126 / science. 1232033, published online 3 January 2013, and through the teachings of this specification, the present invention includes methods and materials of these documents applied in connection with the teachings of this specification. In certain embodiments, on-target binding is increased. In certain embodiments, off-target binding is reduced. In certain embodiments, on-target binding is reduced. In certain embodiments, off-target binding is increased. Thus, the invention also provides to increase or decrease the specificity of on-target binding vs. off-target binding of functional Cas9 binding proteins.

The use of Cas9 as an RNA-guided binding protein is not limited to CRISPR null Cas9. Cas9 enzymes with nuclease activity can also function as RNA-guided binding proteins when used with specific guide RNAs. For example, small guide RNAs and guide RNAs containing nucleotides that are mismatched with the target can promote Cas9 binding to the target sequence, with little or no target cleavage (eg, Dahlman, 2015, Nat). Biotechnology.33 (11): 1159-1161, doi: 10.1038 / nbt.3390, published online 05 October 2015). In some embodiments, the invention provides methods and mutations that regulate the binding of Cas9 proteins, including nuclease activity. In certain embodiments, on-target binding is increased. In certain embodiments, off-target binding is reduced. In certain embodiments, on-target binding is reduced. In certain embodiments, off-target binding is increased. In certain embodiments, there is an increase or decrease in the specificity of on-target binding vs. off-target binding. In certain embodiments, the nuclease activity of the guide RNA-Cas9 enzyme is also regulated.

RNA-DNA heteroduplex formation is important for cleavage activity and specificity across the target region, not just the seed region sequence closest to PAM. Therefore, truncated guide RNAs exhibit reduced cleavage activity and specificity. In certain embodiments, the present invention provides methods and mutations that use altered guide RNAs to increase cleavage activity and specificity.

The present invention also demonstrates that modifications of Cas9 nuclease specificity can be made in line with modifications to the target range. For example, by selecting mutations that alter PAM specificity and combining those mutations with nt groove mutations that increase (or decrease) the specificity of the on-target sequence vs. off-target sequence. Cas9 mutants can be designed that have increased target specificity as well as regulatory alterations in PAM recognition. In one such embodiment, the PI domain residue is mutated to regulate the recognition of the desired PAM sequence, while at the same time mutating one or more nt groove amino acids to alter the target specificity. Kleinstiber is working on SpCas9 and SaCas9 nucleases in which specific PI domain residues are mutated to recognize selective PAM sequences (Kleinstiver et al., Nature 523 (7561): 481-5 doi: 10.1038 /. Nature 14592, published online 22 June 2015; Kleinstiber et al., Nature Biotechnology, doi: 10.1038 / nbt. 3404, published online 2 November 2015). The Cas9 methods and modifications described herein counteract the loss of specificity caused by altered PAM perception, enhance the acquisition of specificity caused by altered PAM perception, and the specificity caused by altered PAM perception. It can counteract the acquisition of sex or enhance the loss of specificity caused by altered PAM perception.

The method and mutations can be used with any Cas9 enzyme with altered PAM recognition. Non-limiting examples of PAM included NGG, NNGRRT, NN [A / C / T] RRT, NGAN, NGCG, NGAG, NGNG, NGC, and NGA.

In a further embodiment, the method and mutations are used with modified proteins.

In any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme can include one or more heterologous functional domains.

One or more heterologous functional domains may include one or more nuclear localization signal (NLS) domains. One or more heterologous functional domains may include at least two or more NLS.

In certain embodiments of the invention, at least one nuclear localization signal (NLS) is added to the nucleic acid sequence encoding the Cas9 effector protein. In a preferred embodiment, at least one or more C-terminal or N-terminal NLS can be added (and thus one or more nucleic acid molecules encoding the Cas9 effector protein can include coding of one or more NLS. The expressed product will have its NLS added or linked). In a preferred embodiment, C-terminal NLS is added for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells. In a preferred embodiment, the codon-optimized effector protein is SpCas9 or SaCas9, and the spacer length of the guide RNA is 15-35 nt. In certain embodiments, the spacer length of the guide RNA is at least 16 nucleotides, eg, at least 17 nucleotides. In certain embodiments, the spacer lengths are 15-17 nt, 17-20 nt, 20-24 nt, eg 20, 21, 22, 23, or 24 nt, 23-25 nt, eg 23, 24, or 25 nt, 24- 27nt, 27-30nt, 30-35nt, or 35nt or more. In certain embodiments of the invention, the codon-optimized effector protein is SpCas9 or SaCas9, and the direct repeat length of the guide RNA is at least 16 nucleotides. In certain embodiments, the codon-optimized effector protein is FnCpf1p and the direct repeat length of the guide RNA is 16-20 nt, eg, 16, 17, 18, 19, or 20 nucleotides. In certain preferred embodiments, the guide RNA has a direct repeat length of 19 nucleotides.

One or more heterologous functional domains include one or more transcription activation domains. The transcriptional activation domain may include VP64.

One or more heterologous functional domains include one or more transcriptional repression domains. The transcriptional repression domain may include a KRAB domain or a SID domain.

One or more heterologous functional domains may include one or more nuclease domains. One or more nuclease domains may include Fok1.

One or more heterologous functional domains can have one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity.

At least one or more heterologous functional domains can be at or near the amino terminus of the enzyme and / or near the carboxy terminus of the enzyme or near it.

One or more heterologous functional domains may be fused to the CRISPR enzyme, moored to the CRISPR enzyme, or linked to the CRISPR enzyme by a linker moiety.

Among any of the non-naturally occurring CRISPR enzymes, the CRISPR enzymes include Streptococcus, Campylobacter, Nitratifragtor, Staphylococcus, and Pulvicula. , Rosebria, Neisseria, Gluconacetobacter, Azospirillum, Spherochaeta, Sphaerochaeta, Lactobacillus, Lactobacillus, Lactobacillus, Lactobacillus. CRISPR enzymes from organisms of the genus including (Corynebacter) may be included.

In any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme includes a chimeric Cas9 enzyme containing a first fragment from a first Cas9 ortholog and a second fragment from a second Cas9 ortholog. Also, the first and second Cas9 orthologs are different. At least one of the first and second Cas9 orthologs includes the genus Streptococcus, the genus Campylobacter, the genus Nitratifractor, the genus Staphylococcus, the genus Staphylococcus, the genus Pulvibaccus. (Rosebulia), Neisseria, Gluconacetobacter, Azospiryllum, Sphaerochaeta, Lactobacillus, Lactobacillus, Ubacillus, Staphylococcus, Staphylococcus. Organized Cas9 may be included.

In any of the non-naturally occurring CRISPR enzymes, the nucleotide sequence encoding the CRISPR enzyme may be codon-optimized for expression in eukaryotes.

In any of the non-naturally occurring CRISPR enzymes, the cell may be a eukaryotic cell or a prokaryotic cell; where the CRISPR complex is operable in the cell, whereby the enzyme in the CRISPR complex is an unmodified enzyme. The ability of the cell to modify one or more off-target loci is reduced when compared to, and / or thereby the enzyme in the CRISPR complex modifies one or more target loci when compared to an unmodified enzyme. Ability increases.

The invention also provides a non-naturally engineered composition comprising a CRISPR-Cas complex comprising any of the non-naturally occurring CRISPR enzymes described above.

The present invention also
A delivery system operably configured to deliver a CRISPR-Cas complex component or one or more polynucleotide sequences containing or encoding the CRISPR-Cas complex to a cell, the CRISPR-Cas complex. The body also provides non-naturally engineered compositions, including delivery systems, that are operable in cells.
A CRISPR-Cas complex component or one or more polynucleotide sequences encoding a CRISPR-Cas complex component for transcription and / or translation in a cell.
(I) The non-naturally occurring CRISPR enzyme according to any one of the preceding claims;
(II) CRISPR-Cas complex RNA comprising:
Guide array,
tracr mate sequence, and tracr sequence,
Including here:
In cells:
The tracr mate sequence hybridizes to the tracr sequence;
A CRISPR complex is formed;
The guide RNA targets the target polynucleotide locus, the enzyme alters the polynucleotide locus, and the enzyme in the CRISPR complex has the ability to modify one or more off-target loci when compared to the unmodified enzyme. It decreases and / or thereby increases the ability of the enzyme in the CRISPR complex to modify one or more target loci when compared to the unmodified enzyme.

In any such composition, the delivery system may include yeast system, lipofection system, microinjection system, microscopic gun system, virosomes, liposomes, immunoliposomes, polycations, lipids: nucleic acid conjugates or artificial virions.

In any such composition, the delivery system may include a vector system comprising one or more vectors, where component (II) is a poly comprising a guide sequence, a tracr mate sequence and a tracr sequence. It comprises a first regulatory element operably linked to a nucleotide sequence, and component (I) comprises a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme. In such compositions, the guide RNA or CRISPR-Cas complex RNA may include a chimeric RNA.

In any such composition, the delivery system may include a vector system comprising one or more vectors, where component (II) is operably linked to a guide sequence and a tracr mate sequence. A second regulatory element comprising a first regulatory element and a third regulatory element operably linked to the tracr sequence, and component (I) operably linked to a polynucleotide sequence encoding a CRISPR enzyme. Includes adjustment elements for.

In any such composition, the composition can include two or more guide RNAs, each guide RNA having a different target, thereby multiplexing.

In any such composition, one or more polynucleotide sequences may be on one vector.

The present invention also
a) A first regulatory element operably linked to a nucleotide sequence encoding a non-naturally occurring CRISPR enzyme in any one of the constructs of the invention herein; and b) encoding one or more of guide RNAs. A second regulatory element operably linked to one or more nucleotide sequences, wherein the guide RNA comprises a guide sequence, a tracr sequence, and a tracr mate sequence.
We also provide an engineered, non-naturally occurring, clustered, regularly spaced short palindrome repeat (CRISPR) -CRISPR-related (cas) (CRISPR-Cas) vector system containing one or more vectors containing so:
Components (a) and (b) are located on the same or different vectors and
The tracr mate sequence hybridizes to the tracr sequence;
A CRISPR complex is formed;
Guide RNA targets the target polynucleotide locus, the enzyme alters the polynucleotide locus, and the enzyme in the CRISPR complex has a reduced ability to modify one or more off-target loci when compared to an unmodified enzyme. And / or thereby increase the ability of the enzyme in the CRISPR complex to modify one or more target loci when compared to the unmodified enzyme.

In such a system, component (II) can include a first regulatory element operably linked to a polynucleotide sequence comprising a guide sequence, a tracr mate sequence and a tracr sequence, and component (II). Can include a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme. In such a system, the guide RNA may include a chimeric RNA.

In such a system, component (I) may include a first regulatory element operably linked to the guide and tracr mate sequences and a third regulatory element operably linked to the tracr sequence. And component (II) can include a second regulatory element operably linked to the polynucleotide sequence encoding the CRISPR enzyme. Such systems can contain more than one guide RNA, each guide RNA having a different target, thereby multiplexing. The components (a) and (b) may be on the same vector.

In any such system comprising a vector, the one or more vectors may include one or more viral vectors, such as one or more retroviruses, lentiviruses, adenoviruses, adeno-associated viruses or simple herpesviruses.

In any such system comprising a regulatory element, at least one of the regulatory elements can include a tissue-specific promoter. Tissue-specific promoters can induce expression in mammalian blood cells, mammalian hepatocytes or mammalian eyes.

In any of the compositions or systems described above, the tracr sequence can include one or more protein-interacting RNA aptamers. One or more aptamers may be located in the tetraloop and / or stemloop 2 of the tracr sequence. One or more aptamers may have the ability to bind to the MS2 bacteriophage coat protein.

In any of the compositions or systems described above, the tracr sequence may be 30 nucleotides or longer in length.

In any of the compositions or systems described above, the cell may be a eukaryotic cell or a prokaryotic cell; where the CRISPR complex is operable in the cell, whereby the enzyme in the CRISPR complex is an unmodified enzyme. The ability of the cell to modify one or more off-target loci is reduced when compared to, and / or thereby the enzyme in the CRISPR complex modifies one or more target loci when compared to an unmodified enzyme. Ability increases.

The invention also provides a CRISPR complex from any of the compositions described above or any of the systems described above.

The invention also provides the engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention that are used therapeutically.

The invention also provides a method of modifying a locus of interest in a cell, the method of contacting the cell with any of the compositions described above or any of the systems described above. Containing, or here the cell, comprises any of the above-mentioned CRISPR complexes present within the cell. In such a method, the cell may be a eukaryotic cell. In such a method, the organism may contain cells. In such a method, the organism does not have to be human or other animal. The invention also provides the engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention that are used to modify loci of interest in cells. The modification preferably comprises contacting the cells with any of the compositions described above or any of the systems described above. The invention also provides the use of engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention in the preparation of a medicament for modifying an intracellular locus of interest. ..

Any such method may be exobibo or in vitro.

Any such method, said modification, may include regulating gene expression. The regulation of gene expression may include activating gene expression and / or suppressing gene expression.

The invention also provides the engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention that are used to modify loci of interest in cells. The invention also provides the use of engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention in the preparation of a medicament for modifying an intracellular locus of interest. .. The modification preferably comprises contacting the cells with any of the compositions described above or any of the systems described above. The invention also provides a method of treating a disease, disorder or infection in an individual in need thereof, wherein the method administers an effective amount of any of the compositions, systems or CRISPR complexes described above. Includes steps to do. Diseases, disorders or infections can include viral infections. The viral infection may be HBV.

The invention also provides the engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention used in the treatment of diseases, disorders or infections of individuals in need thereof. Diseases, disorders or infections can include viral infections. The viral infection may be HBV. The invention also relates to the engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention in the preparation of a medicament for treating a disease, disorder or infection in an individual in need thereof. Also provides the use of. Diseases, disorders or infections can include viral infections.

The invention also provides for the use of any of the compositions, systems or CRISPR complexes described above for gene or genome editing.

The present invention also provides any of the compositions, systems or CRISPR complexes described above for use as therapeutic agents. The therapeutic agent may be for gene or genome editing, or for gene therapy.

In one aspect, the invention is by manipulating a target sequence at a genomic locus of interest in the HSC, eg, the genomic locus of interest is associated with aberrant protein expression or mutation associated with a disease condition or condition. Provides a method of modifying an organism or non-human organism, which method
I. A CRISPR-Cas-based chimeric RNA (chiRNA) polynucleotide sequence
(A) A guide sequence capable of hybridizing to a target sequence in HSC,
(B) tracr mate sequence, and (c) tracr sequence,
Polynucleotide sequence containing, and II. A CRISPR enzyme, optionally containing at least one or more nuclear localization sequences.
Including the step of delivering a non-naturally occurring or engineered composition comprising, eg, by contacting the HSC with particles containing it, to the HSC.
Here the tracr mate sequence hybridizes to the tracr sequence, the guide sequence leads to sequence-specific binding of the CRISPR complex to the target sequence, where the CRISPR complex is (1) a guide sequence that hybridizes to the target sequence. , And (2) a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to the tracr sequence; and this method also optionally, for example, via particles in contact with the HSC containing the HDR template. Alternatively, contacting the HSC with another particle containing the HDR template can also include the step of delivering the HDR template, where the HDR template results in the expression of normal or slightly aberrant proteins; "Normal" is for the wild type, and "abnormal" may be protein expression that causes the condition or disease state; and optionally, the method isolates or obtains HSCs from living or non-human organisms. Steps, optionally expanding the HSC population, performing contact with one or more particles with the HSC to obtain a modified HSC population, optionally expanding the modified HSC population It may include a step and optionally administering the modified HSC to an organism or non-human organism.

The invention also relates to engineered CRISPR proteins, complexes, compositions, systems, vectors, cells according to the invention used to modify an organism or non-human organism by manipulating a target sequence at the genomic locus of interest in HSC. Alternatively, a cell line is also provided. The modification is preferably
I. A CRISPR-Cas-based chimeric RNA (chiRNA) polynucleotide sequence
(A) A guide sequence capable of hybridizing to a target sequence in HSC,
(B) tracr mate sequence, and (c) tracr sequence,
Polynucleotide sequence containing, and II. A CRISPR enzyme, optionally containing at least one or more nuclear localization sequences.
Including the step of delivering a non-naturally occurring or engineered composition comprising, eg, by contacting the HSC with particles containing it, to the HSC.
Here, the tracr mate sequence hybridizes to the tracr sequence, the guide sequence leads to sequence-specific binding of the CRISPR complex to the target sequence, and where the CRISPR complex is (1) a guide sequence that hybridizes to the target sequence. , And (2) a tracr mate sequence that hybridizes to the tracr sequence, and a CRISPR enzyme complexed with the tracr mate sequence. The modification further comprises, optionally, delivering the HDR template via, for example, a particle in contact with the HSC containing the HDR template or by contacting the HSC with another particle containing the HDR template. Here, the HDR template results in the expression of normal or mildly aberrant proteins; "normal" is for the wild type, and "abnormal" may be protein expression that causes a pathological or disease state. The modification is further modified by optionally isolating or obtaining HSC from an organism or non-human organism, optionally expanding the HSC population, and contacting the HSC with one or more particles. It comprises obtaining a populated HSC population, expanding the population of optionally modified HSCs, and administering the optionally modified HSCs to an organism or non-human organism.

In one aspect, the invention is by manipulating a target sequence at a genomic locus of interest in the HSC, eg, the genomic locus of interest is associated with aberrant protein expression or mutation associated with a disease condition or condition. Provided is a method of modifying an organism or a non-human organism, the method of which is described in I. (A) a guide sequence capable of hybridizing to a target sequence in HSC, and (b) at least one or more tracr mate sequences, II. CRISPR enzymes, which optionally have one or more NLS, and III. The tracr mate sequence comprises the step of delivering a non-naturally occurring or engineered composition comprising a polynucleotide sequence comprising the tracr sequence to the HSC, eg, by contacting the particles containing it with the HSC. Hybridizing to the sequence, the guide sequence leads to sequence-specific binding of the CRISPR complex to the target sequence, where the CRISPR complex is (1) the guide sequence that hybridizes to the target sequence, and (2) the tracr sequence. Includes a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to; and this method also optionally, for example, via particles in contact with an HSC containing an HDR template or containing an HDR template. By contacting the HSC with another particle, the HDR template can also be delivered, where the HDR template results in the expression of normal or mildly aberrant proteins; "normal" refers to the wild form. And "abnormality" may be protein expression that causes the condition or disease state; and optionally this method is the step of isolating or obtaining HSCs from living or non-human organisms, optionally HSCs. A step of expanding the population, a step of contacting one or more particles with the HSC to obtain a modified HSC population, an optional step of expanding the modified HSC population, and an optional modification. It may include the step of administering the HSC to a living or non-human organism. The invention also presents organisms or non-organisms by manipulating target sequences at the genomic locus of interest in HSCs (eg, the genomic locus of interest is associated with abnormal protein expression or mutations associated with disease conditions or conditions). Also provided are engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention used in such modifications of human organisms.

This delivery is, for example, any one of the CRISPR complexes via one or more particles containing a vector containing one or more polynucleotides that are functionally linked to one or more regulatory elements. Delivery of one or more polynucleotides encoding the above or all, preferably linked to one or more regulatory elements for in vivo expression. Part or all of the polynucleotide sequence, guide sequence, tracr mate sequence or tracr sequence encoding the CRISPR enzyme may be RNA. If a polynucleotide that is RNA and is said to "contain" the characteristics of such tracr mate sequence is mentioned, it will be understood that the RNA sequence has that characteristic. If the polynucleotide is DNA and is said to contain the characteristics of such tracr mate sequence, the DNA sequence can be transcribed or transcribed into RNA containing the characteristics of the problem. If the feature is a protein such as a CRISPR enzyme, the DNA or RNA sequence referred to can be translated or translated (in the case of DNA, after being first transcribed).

In certain embodiments, the present invention is a genomic locus of interest comprising the step of delivering a non-naturally occurring or engineered composition, eg, by contacting the HSC, eg, abnormal protein expression or disease conditions. Alternatively, the composition provides a method of modifying an organism, such as a mammal including humans or a non-human mammal or organism, by manipulating a target sequence at the genomic locus of interest of the HSC associated with a state-related mutation. Includes one or more particles containing one or more viral, plasmid or nucleic acid molecular vectors (eg, RNA) that functionally encode the composition to express the composition, the composition of which is (A). I. A first regulatory element that is functionally linked to a CRISPR-Cas-based chimeric RNA (chiRNA) polynucleotide sequence, a guide by which the polynucleotide sequence can (a) hybridize to a target sequence in eukaryotic cells. A first regulatory element comprising a sequence, (b) tracr mate sequence, and (c) tracr sequence, and II. An enzyme coding sequence encoding a CRISPR enzyme that comprises at least one or more nuclear localization sequences (or at least one or more nuclear localization sequences, optionally because NLS may not be involved in some embodiments). The second regulatory elements [(a), (b) and (c) functionally linked to are aligned in the direction from 5'to 3', and the constituents I and II are in the same or different vectors of the system. When located and transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex becomes (1) the target sequence. Containing a guide sequence that hybridizes and (2) a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to a tracr sequence], or (B) a non-naturally occurring or engineered composition, I. .. (A) a guide sequence capable of hybridizing to a target sequence in eukaryotic cells, and (b) a first regulatory element operably linked to at least one or more tracr mate sequences, II. A second regulatory element that is functionally linked to the enzyme coding sequence that encodes the CRISPR enzyme, and III. A third regulatory element operably linked to the tracr sequence [Components I, II and III are located in the same or different vectors of the system, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence and The guide sequence induces a sequence-specific binding between the CRISPR complex and the target sequence, and the CRISPR complex is (1) a guide sequence that hybridizes to the target sequence, and (2) a tracr mate sequence that hybridizes to the tracr sequence. Containing a composition comprising a vector system comprising one or more vectors comprising CRISPR enzyme complexed with; the method optionally comprises particles in contact with the HSC containing, for example, an HDR template. A step of delivering the HDR template can also be included, either through or by contacting the HSC with another particle containing the HDR template, where the HDR template expresses normal or slightly aberrant proteins. Bringing; "normal" is for the wild type, and "abnormal" may be protein expression that causes the condition or disease state; and optionally this method isolates HSCs from living or non-human organisms or Obtaining step, optionally expanding the HSC population, optionally contacting one or more particles with the HSC to obtain the modified HSC population, optionally expanding the modified HSC population And optionally include the step of administering the modified HSC to a living or non-human organism. In some embodiments, the components I, II and III are located in the same vector. In other embodiments, components I and II are located in the same vector, while component III is located in another vector. In other embodiments, components I and III are located in the same vector, while component II is located in a different vector. In other embodiments, components II and III are located in the same vector, while component I is located in a different vector. In other embodiments, components I, II and III are each located in a different vector. The present invention also provides a viral or plasmid vector system as described herein. The invention also comprises the step of delivering a non-naturally occurring or engineered composition, eg, by contacting it with an HSC, for example, at a genomic locus of interest in the HSC (eg, the genomic locus of interest is an abnormal protein expression. Engineered according to the invention used in such modifications of an organism, eg, a human or non-human mammal or a mammal including an organism, by manipulating a target sequence (or associated with a mutation associated with a disease condition or condition). CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines are also provided.

Manipulating the target sequence also means that the applicants have epigenetic manipulation of the target sequence. This is due to alteration of the methylation state of the target sequence (ie, methylation or methylation pattern or addition or removal of CpG islands), histone modification, increased or decreased accessibility to the target sequence, or three-dimensional folding. It may be an manipulation of the chromatin state of the target sequence, such as by promoting. If a method of modifying an organism or mammal, including a human, or a non-human mammal or organism by manipulating a target sequence at a genomic locus of interest is mentioned, this may apply to the organism (or mammal) as a whole. , Or (if the organism is a multicellular organism), it will be appreciated that it may be applied only to a single cell or cell population of the organism. For example, in the case of humans, Applicants specifically envision single cells or cell populations, which are preferably modified ex vivo and then reintroduced. In this case, a biopsy or other tissue sample or biofluid sample may be required. Stem cells are also particularly preferred in this regard. However, of course, in vivo embodiments are also envisioned. And the present invention is particularly advantageous with respect to HSC.

The present invention, in some embodiments, for example,
I. The first CRISPR-Cas-based chimeric RNA (chiRNA) polynucleotide sequence.
(A) A first guide sequence capable of hybridizing to a first target sequence,
(B) a first tracr mate sequence, and (c) a first polynucleotide sequence comprising a first tracr sequence,
II. A second CRISPR-Cas-based chiRNA polynucleotide sequence,
(A) A second guide sequence capable of hybridizing to a second target sequence,
(B) A second tracr mate sequence, and (c) a second polynucleotide sequence containing a second tracr sequence, and III. Polynucleotide sequences [(a), (b) and (c) encoding a CRISPR enzyme containing at least one or more nuclear localization sequence and containing one or more mutations from 5'to 3'. Line up in the direction]; or IV. I. ~ III. One or more expression products of, eg, first and second tracr mate sequences, CRISPR enzymes;
[When transcribed, the first and second tracr mate sequences hybridize to the first and second tracr sequences, respectively, and the first and second guide sequences are the first and second CRISPR complexes, respectively. The first guide sequence, which induces sequence-specific binding between the enzyme and the first and second target sequences, and the first CRISPR complex hybridizes to (1) the first target sequence, and (2) the first. A second guide sequence comprising a CRISPR enzyme complexed with a first tracr mate sequence that hybridizes to one tracr sequence, the second CRISPR complex (1) hybridizing to a second target sequence. , And (2) the CRISPR enzyme complexed with the second tracr mate sequence that hybridizes to the second tracr sequence, and the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA, and the first. The guide sequence induces cleavage of one strand of the DNA duplex in the vicinity of the first target sequence, and the second guide sequence induces cleavage of the other strand in the vicinity of the second target sequence. The purpose of the HSC, which comprises the step of delivering by contacting the HSC with particles containing a non-naturally occurring or engineered composition comprising causing a double-strand break thereby altering an organism or non-human organism. Manipulation of first and second target sequences at the genomic locus, eg, on the reverse strand of the DNA duplex at the genomic locus of interest associated with abnormal protein expression or mutations associated with disease conditions or conditions. Includes a method of modifying an organism or non-human organism by; and this method (mhod) is optionally, for example, via particles in contact with an HSC containing an HDR template or separately containing an HDR template. The step of delivering the HDR template can also be included by contacting the particles with the HSC, where the HDR template results in the expression of normal or slightly aberrant proteins; "normal" is for the wild form. And "abnormality" may be protein expression that causes the condition or disease state; and optionally this method is the step of isolating or obtaining HSC from an organism or non-human organism, optionally this HSC population. To obtain a modified HSC population by performing contact with one or more particles of the HSC, including optionally expanding the modified HSC population, and optionally modifying the modified HSC. Steps to administer to living or non-human organisms A method comprising manipulating a target sequence within a coding element, non-coding element or regulatory element of said genomic locus, including. In some methods of the invention, the polynucleotide sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences or some or all of the first and second tracr sequences. Is RNA. In a further embodiment of the invention, the sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences or the polynucleotide encoding the first and second tracr sequences is RNA. There are, delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; however, delivery is advantageous by particles. In certain embodiments of the invention, the first and second tracr mate sequences share 100% identity and / or the first and second tracr sequences share 100% identity. In some embodiments, the polynucleotide can be contained within a vector system that includes one or more vectors. In a preferred embodiment of the invention, the CRISPR enzyme is a Cas9 enzyme, such as SpCas9. In aspects of the invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, the one or more mutations consisting of the group consisting of D10A, E762A, H840A, N854A, N863A and D986A relative to SpCas9. Selected, eg D10A mutation. In a preferred embodiment, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme, and the second CRISPR enzyme is that the enzyme is a non-complementary strand nicking enzyme. Has one or more mutations such as. Alternatively, the first enzyme may be a non-complementary strand nicking enzyme, and the second enzyme may be a complementary strand nicking enzyme. In the preferred method of the invention, the first guide sequence induces cleavage of one strand of the DNA duplex in the vicinity of the first target sequence, and the second guide sequence is in the vicinity of the second target sequence. Inducing breakage of the other strand results in a 5'overhang. In embodiments of the invention, the 5'overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention, the 5'overhang is at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs. The invention also comprises the step of delivering one or more particles containing a non-naturally occurring or engineered composition, eg, by contacting the HSC, for a genomic locus of interest in the HSC (eg, for this purpose). Genomic loci are associated with abnormal protein expression or mutations associated with disease conditions or conditions) of organisms or non-human organisms by manipulating the first and second target sequences on the reverse strand of the DNA duplex. Also provided are engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention used in such modifications.

The present invention, in some embodiments, for example,
I. The first adjustment element
A first guide sequence capable of hybridizing to a first target sequence, and (b) a first regulatory element that is functionally linked to at least one or more tracr mate sequences.
II. The second adjustment element
(A) a second guide sequence capable of hybridizing to a second target sequence, and (b) a second regulatory element that is functionally linked to at least one or more tracr mate sequences.
III. A third regulatory element that is functionally linked to the enzyme coding sequence that encodes the CRISPR enzyme, and IV. A fourth regulatory element, functionally linked to the tracr sequence,
V. I. ~ IV. One or more expression products of, eg, first and second tracr mate sequences, CRISPR enzymes;
[When components I, II, III and IV are located in the same or different vectors of the system and transcribed, the tracr mate sequence hybridizes to the tracr sequence and the first and second guide sequences are first, respectively. And a first guide sequence that induces sequence-specific binding of the first and second CRISPR complexes to the second target sequence and the first CRISPR complex hybridizes to (1) the first target sequence. , And (2) a second guide sequence that contains a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to the tracr sequence, and the second CRISPR complex hybridizes to (1) a second target sequence. , And (2) a polynucleotide sequence comprising a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to the tracr sequence, encoding the CRISPR enzyme is DNA or RNA, and the first guide sequence is the first. Induces cleavage of one strand of the DNA duplex in the vicinity of the target sequence, and the second guide sequence induces cleavage of the other strand in the vicinity of the second target sequence, resulting in double-strand breaks. , Thereby modifying an organism or non-human organism], including the step of delivering one or more particles containing a non-naturally occurring or engineered composition by contacting the HSC with the genomic gene of interest of the HSC. By manipulating the first and second target sequences on the reverse strand of the DNA duplex at the locus, the genomic locus of interest associated with abnormal protein expression or mutations associated with disease conditions or conditions, or Includes methods of modifying non-human organisms; and this method optionally, for example, via a particle that contacts the HSC containing the HDR template, or by contacting the HSC with another particle that contains the HDR template. Thereby, the step of delivering the HDR template can also be included, where the HDR template results in the expression of a normal or slightly aberrant protein; "normal" is for the wild form and "abnormal". May be protein expression that causes a pathological or disease state; and optionally this method is the step of isolating or obtaining HSCs from living or non-human organisms, optionally the step of expanding this HSC population, one. A step of obtaining a modified HSC population by performing contact between the above particles and HSC, a step of optionally expanding the modified HSC population, and a step of optionally administering the modified HSC to an organism or a non-human organism. Including Can be seen. The invention also comprises the step of delivering one or more particles containing a non-naturally occurring or engineered composition, eg, by contacting the HSC, for a genomic locus of interest in the HSC (eg, for this purpose). Genomic loci are associated with abnormal protein expression or mutations associated with disease conditions or conditions) of organisms or non-human organisms by manipulating the first and second target sequences on the reverse strand of the DNA duplex. Also provided are engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention used in such modifications.

The present invention also provides a vector system as described herein. This system may contain one, two, three or four different vectors. Thus, components I, II, III and IV may be located in one, two, three or four different vectors, and all possible combinations of components are envisioned herein. For example: components I, II, III and IV may be located in the same vector in all possible combinations of positions; components I, II, III and IV may be located in different vectors. The components I, II, III and IV may be located in a total of two or three different vectors, etc. In some methods of the invention, the polynucleotide sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences or some or all of the first and second tracr sequences. Is RNA. In a further embodiment of the invention, the first and second tracr mate sequences share 100% identity and / or the first and second tracr sequences share 100% identity. In a preferred embodiment of the invention, the CRISPR enzyme is a Cas9 enzyme, such as SpCas9. In aspects of the invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, the one or more mutations consisting of the group consisting of D10A, E762A, H840A, N854A, N863A and D986A relative to SpCas9. Selected; eg D10A mutation. In a preferred embodiment, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme, and the second CRISPR enzyme is that the enzyme is a non-complementary strand nicking enzyme. Has one or more mutations such as. Alternatively, the first enzyme may be a non-complementary strand nicking enzyme, and the second enzyme may be a complementary strand nicking enzyme. In a further embodiment of the invention, one or more of the viral vectors are delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; however, particle delivery is advantageous.

In the preferred method of the invention, the first guide sequence induces cleavage of one strand of the DNA duplex in the vicinity of the first target sequence, and the second guide sequence is in the vicinity of the second target sequence. Inducing breakage of the other strand results in a 5'overhang. In embodiments of the invention, the 5'overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention, the 5'overhang is at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs.

The present invention, in some embodiments, comprises, for example, a Cas protein having one or more mutations and two guide RNAs that target the first and second strands of the DNA molecule in the HSC, respectively. By contacting the containing particles with the HSC, they are introduced into the HSC, whereby the guide RNA targets the DNA molecule and the Cas protein nicks each of the first and second strands of the DNA molecule, thereby. By altering the target in the HSC (and the Cas protein and the two guide RNAs do not coexist in nature), the genomic locus of interest in the HSC, eg, abnormal protein expression or disease conditions or conditions. Includes a method of modifying a genomic locus of interest associated with a related mutation, and this method (protein) is optionally, for example, via a particle in contact with an HSC containing an HDR template or HDR. By contacting the HSC with another particle containing the template, the step of delivering the HDR template can also be included, where the HDR template results in the expression of a normal or slightly aberrant protein; "normal". Is related to the wild form, and "abnormality" may be protein expression that causes the condition or disease state; and optionally this method is the step of isolating or obtaining HSCs from living or non-human organisms, optionally. A step of optionally expanding this HSC population, a step of contacting one or more particles with the HSC to obtain a modified HSC population, an optional step of expanding the modified HSC population, and an optional modification. It may include the step of administering HSC to a living or non-human organism. In the preferred method of the invention, the Cas protein nicks each of the first and second strands of the DNA molecule, resulting in a 5'overhang. In embodiments of the invention, the 5'overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention, the 5'overhang is at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs. Embodiments of the present invention also include a guide sequence fused to a tracr mate sequence and a guide RNA containing the tracr sequence. In aspects of the invention, the Cas protein is codon-optimized for expression in eukaryotic cells, preferably mammalian or human cells. In a further embodiment of the invention, the Cas protein is a type II CRISPR-Cas protein, such as the Cas9 protein. In a highly preferred embodiment, the Cas protein is a Cas9 protein, such as SpCas9 or SaCas9. In aspects of the invention, the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A based on SpCas9; eg, D10A mutations. Aspects of the invention allow the expression of the gene product to be reduced, or the template polynucleotide to be further introduced into the DNA molecule encoding the gene product, or the rearnying and ligation of two 5'overhangs. This relates to accurately excising the intervening sequence, or altering the activity or function of the gene product, or increasing the expression of the gene product. In certain embodiments of the invention, the gene product is a protein.

The present invention, in some embodiments, for example,
a) A first regulatory element that is functionally linked to each of the two CRISPR-Cas-based guide RNAs that target the first and second strands of the HSC double-stranded DNA molecule, respectively, and b. ) A second regulatory element that is functionally linked to the Cas protein, or one or more expression products of c) a) or b).
One or more particles containing [components (a) and (b) are located in the same or different vectors of the system] are introduced into the HSC by contacting the HSC, whereby the guide RNA is the DNA molecule of the HSC. By targeting and the Cas protein nicking each of the first and second strands of its HSC DNA molecule (and the Cas protein and the two guide RNAs do not exist together in nature). Includes methods of modifying genomic loci of interest in HSCs, such as genomic loci of interest associated with abnormal protein expression or mutations associated with disease conditions or conditions; and this method is optional, eg, HDR template. A step of delivering an HDR template can also be included, either via a particle in contact with the HSC containing the HSC or by contacting the HSC with another particle containing the HDR template, where the HDR template is normal or It results in the expression of a low degree of abnormal protein; "normal" may be for the wild type, and "abnormal" may be protein expression that causes the condition or disease state; and optionally this method is biological or Isolation or acquisition of HSCs from non-human organisms, optional expansion of the HSC population, optional contact of one or more particles with the HSC to obtain a modified HSC population, optional It may include expanding the population of modified HSCs and optionally administering the modified HSCs to living or non-human organisms. In aspects of the invention, the guide RNA may comprise a guide sequence and a tracr sequence fused to a tracr mate sequence. In certain embodiments of the invention, the Cas protein is a type II CRISPR-Cas protein. In aspects of the invention, the Cas protein is codon-optimized for expression in eukaryotic cells, preferably mammalian or human cells. In a further embodiment of the invention, the Cas protein is a type II CRISPR-Cas protein, such as the Cas9 protein. In a highly preferred embodiment, the Cas protein is a Cas9 protein, such as SpCas9 or SaCas9. In aspects of the invention, the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A relative to SpCas9; eg, D10A mutations. Aspects of the invention allow the expression of the gene product to be reduced, or the template polynucleotide to be further introduced into the DNA molecule encoding the gene product, or the rearnying and ligation of two 5'overhangs. This relates to accurately excising the intervening sequence, or altering the activity or function of the gene product, or increasing the expression of the gene product. In certain embodiments of the invention, the gene product is a protein. In a preferred embodiment of the invention, the vector of the system is a viral vector. In a further embodiment, the vector of the system is delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; and particles are preferred. In one aspect, the invention provides a method of modifying a target polynucleotide in HSC. In some embodiments, the method comprises binding the CRISPR complex to a target polynucleotide, resulting in cleavage of the target polynucleotide, thereby modifying the target polynucleotide. The CRISPR complex contains a CRISPR enzyme that hybridizes with a guide sequence that hybridizes to the target sequence within the target polynucleotide, the guide sequence is linked to the tracr mate sequence, and then the tracr mate sequence is the tracr sequence. Hybridizes to. In some embodiments, the cleavage comprises cleaving one or two strands at the location of the target sequence with the CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises the step of repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair comprises one or more nucleotides of the target polynucleotide. It results in mutations involving insertions, deletions, or substitutions of. In some embodiments, the mutation results in one or more amino acid changes in the protein expressed from the gene containing the target sequence. In some embodiments, the method further comprises the step of delivering one or more vectors or one or more expression products thereof to the HSC via, for example, one or more particles, wherein one or more. The vector drives the expression of one or more of the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence. In some embodiments, the vector is delivered to the HSC of interest. In some embodiments, the modification step is performed on the HSC in a cell culture. In some embodiments, the method further comprises the step of isolating the HSC from the subject prior to the modification step. In some embodiments, the method further comprises the step of returning the HSC and / or cells derived from it to the subject.

In one aspect, the invention provides a method of making an HSC containing a mutant disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of having or developing the disease. In some embodiments, the method is (a) the step of introducing one or more vectors or one or more expression products thereof into the HSC via, for example, one or more particles, one or more. The vector drives the expression of one or more of the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence; and (b) the step of binding the CRISPR complex to the target polynucleotide, said. It causes cleavage of the target polynucleotide in the disease gene [CRISPR complex is complexed with (1) a guide sequence that hybridizes to the target sequence in the target polynucleotide, and (2) a tracr mate sequence that hybridizes to the tracr sequence. Includes the CRISPR enzyme that formed the body], thereby including the step of creating an HSC containing the mutant disease gene. In some embodiments, the cleavage comprises cleaving one or two strands at the location of the target sequence with the CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises the step of repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair comprises one or more nucleotides of the target polynucleotide. It results in mutations involving insertions, deletions, or substitutions of. In some embodiments, the mutation results in one or more amino acid changes in protein expression from the gene containing the target sequence. In some embodiments, the modified HSC is administered to an animal, thereby creating an animal model.

In one aspect, the invention provides a method of modifying a target polynucleotide in an HSC. Also provided are engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines according to the invention used to modify target polynucleotides within HSCs. In some embodiments, the method comprises binding the CRISPR complex to a target polynucleotide, resulting in cleavage of the target polynucleotide, thereby modifying the target polynucleotide. The body comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to the target sequence within the target polynucleotide, the guide sequence is ligated to the tracr mate sequence, and then the tracr mate sequence hybridizes to the tracr sequence. To soy. In another embodiment, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell resulting from HSC. The method comprises increasing or decreasing the expression of the target polynucleotide using a CRISPR complex that binds to the polynucleotide in the HSC; advantageously the CRISPR complex is delivered via one or more particles. Will be done.

In some methods, inactivation of the target polynucleotide can result in altered expression in the HSC. For example, when the CRISPR complex binds to a target sequence in a cell, the target polynucleotide is inactivated, so that the sequence is not transcribed and no coding protein is produced, or the sequence functions like a wild-type sequence. Will not be.

In some embodiments, RNA of the CRISPR-Cas system, such as a guide or sgRNA, can be modified; eg, an aptamer or functional domain can be included. Aptamers are synthetic oligonucleotides that bind to specific target molecules; repeated rounds of in vitro selection or SELEX to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. It is a nucleic acid molecule engineered by the in vitro evolution method). Aptamers are useful in providing molecular recognition properties that compete with antibodies. In addition to its discriminative recognition, aptamers offer advantages over antibodies, including little or no immunogenicity in therapeutic applications. Thus, in practicing the present invention, one or both of the enzyme or RNA may contain a functional domain.

In some embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is a SID, or a concatemer of the SID (eg, SID4X). In some embodiments, the functional domain is an epigenetic modification domain, thus providing an epigenetic modification enzyme. In some embodiments, the functional domain is the activation domain, which may be the P65 activation domain. In some embodiments, the functional domain comprises nuclease activity. In one such embodiment, the functional domain comprises Fok1.

The present invention also provides in vitro or exobibo cells comprising any of the modified CRISPR enzymes, compositions, systems or complexes described above or by any of the methods described above. This cell may be a eukaryotic cell or a prokaryotic cell. The present invention also provides progeny of such cells. The invention also provides the product of any such cell or any such progeny, which product is the product of the one or more target loci as modified by the modified CRISPR enzyme of the CRISPR complex. The product may be a peptide, polypeptide or protein. Some such products may be modified by a modified CRISPR enzyme of the CRISPR complex. In some such modified products, the product of the target locus is physically different from the product of the target locus that has not been modified by the modified CRISPR enzyme.

The invention also provides a polynucleotide molecule comprising a polynucleotide sequence encoding any of the non-naturally occurring CRISPR enzymes described above.

Any such polynucleotide may further comprise one or more regulatory elements operably linked to a polynucleotide sequence encoding a non-naturally occurring CRISPR enzyme.

In any such polynucleotide containing one or more regulatory elements, one or more regulatory elements may be operably configured for expression of the non-naturally occurring CRISPR enzyme in eukaryotic cells. The eukaryotic cell may be a human cell. Eukaryotic cells may be rodent cells, optionally mouse cells. The eukaryotic cell may be a yeast cell. Eukaryotic cells may be Chinese hamster ovary (CHO) cells. Eukaryotic cells may be insect cells.

In any such polynucleotide containing one or more regulatory elements, one or more regulatory elements may be operably configured for expression of the non-naturally occurring CRISPR enzyme in prokaryotic cells.

In any such polynucleotide containing one or more regulatory elements, one or more regulatory elements may be operably configured for expression of a non-naturally occurring CRISPR enzyme in an in vitro system.

The present invention also provides an expression vector containing any of the above-mentioned polynucleotide molecules. The present invention also comprises one or more such polynucleotide molecules, eg, such polynucleotide molecules operably configured to express one or more protein and / or nucleic acid constituents, and one or more. Such vectors are also provided.

The present invention further provides a method of creating a mutation in Cas9 or a mutated or modified Cas9 that is an ortholog of SaCas9 and / or SpCas9, which is for modification and / or mutation. , One or more amino acids in the ortholog, and / or books in SaCas9 and / or SpCas9, which may be in close proximity to or in contact with nucleic acid molecules such as DNA, RNA, sgRNA, etc. Includes or then includes steps to determine one or more amino acids that are similar or corresponding to one or more amino acids identified in the specification, and one or more modifications and / or one or more mutations. The step of synthesizing, preparing, or expressing an ortholog that becomes or is essentially from it or suddenly charging a neutral amino acid, eg, to a positively charged amino acid, eg, alanine, to, for example, lysine, as discussed herein. Includes steps to mutate, eg modify, eg modify or mutate. The ortholog thus modified can be used in a CRISPR-Cas system; and one or more nucleic acid molecules expressing it can deliver the molecule or have a CRISPR-Cas system configuration as discussed herein. It can be used in vectors encoding components or other delivery systems.

The invention also relates to the engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines, or SaCas9 and / or SpCas9 according to the invention used to create mutations in Cas9. Also provided is a mutated or modified Cas9 that is an ortholog of the, which may be in close proximity to or in proximity to a nucleic acid molecule for modification and / or mutation, such as DNA, RNA, sgRNA. One or more amino acids in the ortholog that may be in contact, and / or one or more amino acids similar or corresponding to the one or more amino acids identified herein in SaCas9 and / or SpCas9. And the step of synthesizing, preparing, or expressing an ortholog containing, consisting of, or essentially consisting of one or more modifications and / or one or more mutations. It comprises the steps of mutating, eg, modifying, eg, altering or mutating a sex amino acid to a charged, eg, positively charged amino acid, eg, alanine, eg, lysine, as discussed herein.

In some embodiments, the present invention provides efficient on-target activity while minimizing off-target activity. In some embodiments, the invention provides efficient on-target cleavage with the CRISPR protein and minimizes off-target cleavage with the CRISPR protein. In some embodiments, the invention provides a guide-specific binding of a CRISPR protein at a locus without DNA breaks. In some embodiments, the invention provides efficient on-target binding guided by a guide of the CRISPR protein at a locus and minimizes off-target binding of the CRISPR protein. Thus, in some embodiments, the invention provides target-specific gene regulation. In some embodiments, the invention provides a guide-specific binding of a CRISPR enzyme at a locus without DNA breaks. Thus, in some embodiments, the present invention provides cleavage at one locus with a single CRISPR enzyme and provides gene regulation at another locus. In some embodiments, the invention provides orthogonal activation and / or inhibition and / or cleavage of multiple targets using one or more CRISPR proteins and / or enzymes.

In another embodiment, the invention provides a method of screening the function of genes in the genome in an exobibo or in vivo cell pool, the method of administration of a library containing multiple CRISPR-Cas-based guide RNAs (sgRNAs) or. Expression involves, where the screening further comprises the use of a CRISPR enzyme, the CRISPR complex is modified to include a heterologous functional domain. In some embodiments, the invention provides a method of screening a genome, including administration of a library to a host or in vivo expression in the host. In some embodiments, the invention provides a method as discussed herein, further comprising an activator administered to or expressed in the host. In some embodiments, the invention provides the method as discussed herein, where the activator is added to the CRISPR protein. In some embodiments, the invention provides the method as discussed herein, where the activator is added to the N-terminus or C-terminus of the CRISPR protein. In some embodiments, the invention provides the method as discussed herein, where the activator is added to the sgRNA loop. In some embodiments, the invention provides a method as discussed herein, further comprising a repressor administered to or expressed in the host. In some embodiments, the invention provides a method as discussed herein, wherein screening involves affecting and detecting gene activation, gene inhibition, or cleavage of a locus. ..

In some embodiments, the invention provides the method as discussed herein, where the host is a eukaryotic cell. In some embodiments, the invention provides the method or use as discussed herein, where the host is a mammalian cell. In some embodiments, the invention provides the method as discussed herein, where the host is a non-human eukaryotic cell. In some embodiments, the invention provides the method as discussed herein, wherein the non-human eukaryotic cell is a non-human mammalian cell. In some embodiments, the invention provides the methods as discussed herein, wherein non-human mammalian cells are, but are not limited to, primates, bovines, sheep, procines, dogs, and others. Rodents, leporidae, such as monkeys, cows, sheep, pigs, dogs, rabbits, rats or mouse cells can be included. In some embodiments, the invention provides the method or use as discussed herein, in which the cells are poultry birds (eg chickens), vertebrate fish (eg salmon) or crustaceans (eg oysters). , Clams, lobsters, shrimp) cells and the like may be non-mammalian eukaryotic cells. In some embodiments, the invention provides the method or use as discussed herein, the non-human eukaryotic cell being a plant cell. The plant cells may be monocotyledonous or dicotyledonous or crop or cereal plants such as cassava, corn, sorghum, soybean, wheat, oat wheat or rice. Plant cells are also algae, trees or producing plants, fruits or vegetables (eg, citrus trees, such as orange, grapefruit or lemon trees; peach or nectarin trees; apples or peppers trees; almonds or walnuts or pistachios. Fruit trees such as trees; Brassica plants; Brassica plants; Lettuces plants; Spinacia plants; Capsicum plants; cotton, tobacco, asparagus Gas, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuces, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa and other trees).

In certain embodiments, the present invention provides the methods as discussed herein comprising delivery of a CRISPR-Cas complex or one or more components thereof or one or more nucleic acid molecules encoding it. And here the one or more nucleic acid molecules are operably linked to one or more regulatory sequences and expressed in vivo. In some embodiments, the invention provides the method as discussed herein, where in vivo expression is by lentivirus, adenovirus, or AAV. In some embodiments, the invention provides the method as discussed herein, where delivery is by particles, nanoparticles, lipids or cell permeable peptides (CPPs).

In some embodiments, the invention provides a pair of CRISPR-Cas complexes, each containing a guide RNA (sgRNA) containing a guide sequence capable of hybridizing to a target sequence at the genomic locus of interest in the cell. At least one loop of each sgRNA is modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and the adapter proteins associate with one or more functional domains and each CRISPR- Each sgRNA of Cas contains a functional domain having DNA cleaving activity. In some embodiments, the invention provides a pair of CRISPR-Cas complexes as discussed herein, where the DNA cleavage activity is due to the Fok1 nuclease.

In some embodiments, the invention comprises a target at a genomic locus of interest, comprising delivering the CRISPR-Cas complex or one or more components thereof or one or more nucleic acid molecules encoding it to a cell. A method for breaking a sequence is provided, wherein the one or more nucleic acid molecules are operably linked to one or more regulatory sequences and expressed in vivo. In some embodiments, the invention provides the method as discussed herein, where delivery is by lentivirus, adenovirus, or AAV. In some embodiments, the invention provides a pair of CRISPR-Cas complexes as discussed herein or as discussed herein, wherein the target of the first complex of the pair. The sequence is on the first strand of the double-stranded DNA, and the target sequence of the second complex of the pair is on the second strand of the double-stranded DNA. In some embodiments, the invention provides a pair of CRISPR-Cas complexes as discussed herein or as discussed herein, wherein the target sequences of the first and second complexes. Because they are close to each other, the DNA is cleaved in a way that promotes homology-dependent repair. In some embodiments, the methods herein can further include the step of introducing template DNA into cells. In some embodiments, the methods herein or the pair of CRISPR-Cas complexes herein are mutated CRISPR enzymes that have less than about 5% of the nuclease activity of the unmutated CRISPR enzymes. It may include having each CRISPR-Cas complex.

In some embodiments, the invention provides a library, method or complex as discussed herein, wherein the sgRNA is modified to have at least one non-coding functional loop, eg, at least one non. Code functional loops are inhibitory; for example, at least one non-code functional loop contains an Alu.

In one aspect, the invention provides a method of altering or modifying the expression of a gene product. The method involves introducing into a cell containing and expressing a DNA molecule encoding a gene product an engineered, non-naturally occurring CRISPR-Cas system containing a Cas protein and a guide RNA that targets the DNA molecule. Can include, thereby the guide RNA targets the DNA molecule encoding the gene product, and the Cas protein cleaves the DNA molecule encoding the gene product, thereby altering the expression of the gene product; and Cas protein and guide RNA do not coexist in nature. The present invention includes a guide RNA containing a guide sequence fused to a tracr sequence. The present invention further includes a Cas protein codon-optimized for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

The invention also relates to engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cells as defined herein used to alter expression of a genomic locus of interest in mammalian cells. We also offer stocks. The invention also provides the use of engineered CRISPR proteins, complexes, compositions, systems, vectors, cells or cell lines for the preparation of drugs that alter the expression of a genomic locus of interest in mammalian cells. The alteration is preferably a step of contacting the cell with the engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention, thereby delivering the vector, and CRISPR. Includes the steps of forming a -Cas complex and binding it to a target. The alteration further preferably comprises the step of determining whether the expression of the genomic locus has been altered.

In some embodiments, the present invention provides altered cells and their progeny, as well as the products produced by those cells. The CRISPR-Cas9 protein and system of the present invention are used to generate cells containing a modified target locus. In some embodiments, the method can include binding the nucleic acid targeting complex to the target DNA or RNA to cause cleavage of the target DNA or RNA, thereby modifying the target DNA or RNA. , Where the nucleic acid targeting complex comprises a nucleic acid targeting effector protein complexed with a guide RNA that hybridizes to the target sequence in the target DNA or RNA. In one aspect, the invention provides a method of repairing a cell locus. In another aspect, the invention provides a method of modifying the expression of DNA or RNA in eukaryotic cells. In some embodiments, the method comprises binding a nucleic acid targeting complex to a DNA or RNA, which causes an increase or decrease in expression of the DNA or RNA; where the nucleic acid targeting complex is present. Includes a nucleic acid targeting effector protein complexed with a guide RNA. Similar considerations and conditions apply as described above to methods of modifying the target DNA or RNA. In practice, these sampling, culturing and reintroduction options apply to all aspects of the invention. In some embodiments, the invention provides a method of modifying a target DNA or RNA of a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal, and modifying one or more cells. Culturing can be done in Exobibo at any stage. Such cells are, without limitation, plant cells, animal cells, specific cell types of any organism, such as stem cells, immune cells, T cells, B cells, dendritic cells, cardiovascular cells, epithelial cells, stem cells and the like. possible. When modified by the present invention, cells can produce, for example, a controlled amount of gene product, which may be increased or decreased depending on the application and / or mutated. In certain embodiments, the loci of the cell are repaired. The one or more cells may be further reintroduced into a non-human animal or plant. For cells to be reintroduced, it may be preferable that the cells are stem cells.

In some embodiments, the present invention provides cells that transiently contain a CRISPR system, or component. For example, when a CRISPR protein or enzyme and nucleic acid is transiently donated to a cell and the locus is altered, the amount of one or more components of the CRISPR system is subsequently reduced. Subsequently, cells that have undergone genetic alterations mediated by CRISPR, progeny of the cells, and organisms containing the cells either contain one or more CRISPR system components in reduced amounts, or one or more of them. It no longer contains system components. One non-limiting example is the self-inactivating CRISPR-Cas system as further described herein. Accordingly, the present invention includes cells and organisms that include one or more loci altered by the CRISPR-Cas system but are essentially deficient in one or more CRISPR system components, as well as cells and progeny of the organism. provide. In certain embodiments, the CRISPR-based components are substantially absent. Such cells, tissues and organisms advantageously contain the desired or selected genetic alterations, but act potentially non-specifically, leading to safety issues or hindering regulatory approval. Potential CRISPR-Cas components or their relics have been lost. In addition, the present invention provides the cells, organisms, and products produced by the cells and their offspring.

The present invention further provides a method of improving the specificity of a CRISPR system by providing the engineered CRISPR protein according to the invention. Preferably, an engineered CRISPR protein, where the protein is complexed with a nucleic acid molecule containing RNA to form a CRISPR complex.
Here, when in the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci,
This protein contains at least one modification compared to the unmodified protein and contains
Here, the activity of the CRISPR complex containing the modified protein is changed when compared with the complex containing the unmodified protein. The at least one modification is preferably in the RuvC and / or HNH domain as described herein, or in the junction groove between the HNH domain and the RuvC domain. A preferred modification is a mutation as described herein.

The invention further provides the use of an engineered CRISPR protein according to the invention to improve the specificity of the CRISPR system, preferably an engineered CRISPR protein, where the protein is complexed with a nucleic acid molecule containing RNA. Embody to form a CRISPR complex,
Here, when in the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci,
Here, the protein is modified to include at least one modification compared to the unmodified protein.
Here, the activity of the CRISPR complex containing the modified protein is changed when compared with the complex containing the unmodified protein. The at least one modification is preferably in the RuvC and / or HNH domain as described herein, or in the junction groove between the HNH domain and the RuvC domain. A preferred modification is a mutation as described herein.

The novel features of the present invention will be described in particular in the appended claims. The features and advantages of the invention will be better understood from the following detailed description and accompanying drawings illustrating exemplary embodiments in which the principles of the invention are utilized.

It is understood that the applicant / inventor is not necessarily bound by any particular theory set forth herein or any particular figure including FIG. 1 by providing a schematic overview. It shall be. This figure considers mutations that improve specificity of positively charged residues that bind to non-target gDNA strands. The data in this schematic summary table are as follows and relate to mutations in SpCas9: Based on SpCas9 numbering, FIG. 1A shows alanine mutations that improve specificity distributed along the groove of the non-targeting strand, such as Arg780, Lys810, Lys855, Lys848, Lys1003, Arg1060, Arg976, His982. Without wishing to be bound by any one particular theory, this mechanism proposal is that the nuclease activity is inactive until the non-target DNA strand sterically causes an HNH conformational change; with HNH. The non-target strand that binds to the groove with RuvC depends on RNA: DNA pairing; mutations in the DNA binding residue in the groove increase the energy requirement for the correct RNA: DNA pairing (Fig. 1B). It is a thing. By using the information herein, including FIG. 1, one of ordinary skill in the art can readily prepare mutants of other Cas9s (eg, other than SpCas9) that exhibit improved or reduced off-target effects. it can. For example, the references cited herein provide information on a number of orthologs for SpCas9 and SaCas9 exemplified herein. From such information, including the sequence information of those other Cas9s, one of ordinary skill in the art will, in addition to SpCas9 and SaCas9 exemplified herein, similar mutants with reduced off-target effects in the Cas9 ortholog. , Can be easily prepared from the information in the present disclosure. In addition, the literature herein provides crystal structure information for Cas9, eg SpCas9; and structural comparisons between crystal structures, eg SpCas9 crystal structure and ortholog crystal structure relative to it, are easy. It can also be done, and thereby, in addition to SpCas9, similar mutants with reduced off-target effects in Cas9 orthologs can be easily obtained without undue experimentation. Therefore, the present invention can be widely applied to modifications or mutations in various Cas9 orthologs to reduce off-target effects, including but not limited to SpCas9 and SaCas9. As further discussed herein, additional or further modifications of the Cas9 enzyme described above can be readily achieved, whereby the enzyme in the CRISPR complex is one or more when compared to the unmodified enzyme. Increased ability to modify target loci.
Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. Shown are 49 point mutants of SpCas9. The target sequence of EMX1.3 is the sequence of the EMX1 gene and its activity is compared with the associated off-target sequence (OT 46). Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. Shown are 49 point mutants of SpCas9. The target sequence is the sequence of the VEGFA gene and its activity is compared with two related off-target sequences (OT 1 and OT 2). Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. The target sequence is the sequence of the VEGFA gene and its activity is compared with three related off-target sequences (OT 1, OT 4, and OT 18). Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. It shows point mutants that show specificity compared to off-target sequences. The target sequence is the sequence of the EMX1 and VEGFA genes, and the activity is compared with nine related off-target sequences. Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. A double mutant of SpCas9 is shown. The target sequence is the sequence of the EMX1 gene and its activity is compared with two related off-target sequences. Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. A double mutant of SpCas9 is shown. The target sequence is the sequence of the VEGFA gene and its activity is compared with three related off-target sequences. Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. Shown are 14 triple mutants of SpCas9. The target sequence is the sequence of the EMX1 and VEGFA genes, and the activity is compared with four related off-target sequences (OT 46, OT 1, OT 4 and OT 18). Shows the activity of the modified SaCas9 enzyme as measured by% indel formation. The target sequence EMX101 is a sequence of the EMX1 gene and compares its activity with three related off-target sequences (OT1, OT2 and OT3). Shows the activity of the modified SaCas9 enzyme as measured by% indel formation. The target sequence of EMX101 is the sequence of EMX1 and its activity is compared with the associated off-target sequence (OT3). The phylogenetic tree of the Cas gene is shown; from the teachings herein and knowledge of the art, the exemplified SpCas9 and SaCas9 mutations or modifications can be applied to other Cas9s. Shown is a phylogenetic analysis that reveals five Cas9 families, including three groups of large Cas9 (about 1400 amino acids) and two groups of small Cas9 (about 1100 amino acids); the teachings herein and the art. From this knowledge, mutations or modifications of the exemplified SpCas9 and SaCas9 can be applied to other Cas9 (thus, the present invention relates to Cas9 in FIG. 9 and Cas9 families and groups herein with respect to SpCas9 and SaCas9. Including modifications or mutations as exemplified). Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. 10A-10C show the target sequences EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1.10, DNMT1.1, DNMT1.2, DNMT1.4, DNMT1.7, VEGFA4, VEGFA5. , And the activity of VEGFA3. FIG. 10D shows VEGFA3 activity compared to off-target sequence OT4. Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. 11A-11C show the target sequences EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1.10, DNMT1.1, DNMT1.2, DNMT1.4, DNMT1.7, VEGFA4, VEGFA5. , And the activity of VEGFA3. FIG. 11D shows VEGFA3 activity compared to off-target sequence OT4. The activity of the modified SpCas9 enzyme (enzymes) as measured by% indel formation is shown. The target sequence is VEGFA3 and its activity is compared with four related off-target sequences (OT1, OT2, OT4 and OT18). The activity of the modified SpCas9 enzyme (enzymes) as measured by% indel formation is shown. The target sequence is VEGFA3 and its activity is compared with four related off-target sequences (OT1, OT2, OT4 and OT18). Shows the activity of the modified SpCas9 enzyme as measured by% indel formation. Shown are 14 point mutants of SpCas9. The target sequence is EMX1.3 and the activity is compared with five related off-target sequences (OT14, OT23, OE35, OT46, and OT53). It shows the structural features and improved specificity of SpCas9. Panel A is a model of target rewinding. The nt groove between the RuvC domain (teal) and the HNH domain (magenta) stabilizes DNA unwinding by non-specific DNA interactions with non-complementary strands. The RNA: cDNA and Cas9: n cDNA interactions compete with cDNA: n cDNA rehybridization (lower arrow) to drive DNA unwinding (upper arrow). Panel B: Structure of SpCas9 (PDB ID 4UN3) showing an nt groove located between the HNH (magenta) and RuvC (teal) domains. A non-target DNA strand (red) was manually modeled in the nt groove (inset). Panel C: Screening for alanine point mutants for improved specificity. Panel D: Evaluation of top point mutants at additional off-target loci. The top five specificity-imparting mutants are highlighted in red. Panel E: Combined mutants improve specificity compared to single point mutants. eSpCas9 (1.0) and eSpCas9 (1.1) are highlighted in red. Panel F: Screening for on-target cleavage efficiency of high-point and combination mutants at 10 target loci. SpCas9 (K855A), eSpCas9 (1.0), and eSpCas9 (1.1) are highlighted in red. It shows the maintenance of on-target efficiency by the spCas9 mutant. Panel A shows an assessment of the on-target cleavage efficiency of SpCas9 mutants when compared to SpCas9 for 24 sgRNAs targeting 9 genomic loci. Panel B is a boxplot of normalized on-target indel formation for mutants SpCas9 (K855A), eSpCas9 (1.0) and eSpCas9 (1.1). Panel C is a Western blot of SpCas9 and mutants using anti-SpCas9 antibody. It shows the susceptibility of spCas9 and mutants K855A, eSpCas9 (1.0), and eSpCas9 (1.1) to single and bibase mismatches between the guide RNA and the target DNA. Panel A shows a mismatched guide sequence for VEGFA targets. Panel B provides a heatmap of spCas9 and the three mutants showing indel% when the guide sequence has a single nucleotide mismatch. Panel C shows indel formation when the guide sequence contains a continuous base conversion mismatch. Compared to wild type: eSpCas9 (1.0) contains K810A, K1003A, R1060A; eSpCas9 (1.1) contains K848A, K1003A, R1060A. Shows an unbiased, genome-wide off-target profile of mutants SpCas9 (K855A) and eSpCas9 (1.1). Panel A is a schematic overview of the BLESS (Direct In situ Cutting Label, Enrichment with Streptavidin and Next Generation Sequencing) workflow. Panel B shows representative BLESS sequencing for genome-mapped forward (red) and reverse (blue) reads. The lead mapping at the Cas9 cleavage site has a characteristic shape compared to the DSB hotspot. Panels C and D show Manhattan plots of genome-wide DSB clusters generated by each SpCas9 mutant using the EMX1 (1) (panel C) and VEGFA (1) (panel D) targeting guides. Panels E and F show targeted deep sequencing validation of off-target sites identified by BLESS. Order off-target sites by DSB score (blue heatmap). The green heatmap shows the sequence similarity between the target sequence and the off-target sequence. A schematic diagram of sgRNA-guided targeting and DNA unwinding is shown. Cas9 cleaves the target DNA through a series of coordinated steps. First, the PAM interaction domain recognizes the NGG sequence on the 5'side of the target DNA. After PAM binding, the first 10-12 nucleotides (seed sequence) of the target sequence are sampled for sgRNA: DNA complementarity, a process that relies on the separation of DNA double strands. If the seed sequence nucleotide is complementary to the sgRNA, the remaining DNA is unwound and the full length of the sgRNA hybridizes to the target DNA strand. In this model, the nt groove between the RuvC domain (teal) and the HNH domain (magenta) stabilizes the non-target DNA strand by non-specific interaction with the positive charge of the DNA phosphate backbone and promotes unwinding. To do. RNA: cDNA and Cas9: n cDNA interactions drive DNA unwinding (up arrow) in competition with cDNA: n cDNA rehybridization (down arrow). The electrostatics of SpCas9 revealing the non-target strand groove are shown. (A) Crystal structure of SpCas9 paired with sgRNA and target DNA colored by electrostatic potential to highlight the positively charged region (4UN3). The scale is -10 to 1 keV. (B) Same as panel (A), except for the HNH domain, which makes the sgRNA: DNA heteroduplex visible. (C) Crystal structure colored for each domain (same orientation as (A)): HNH (magenta), RuvC (teal), and PAM interaction (PI) (beige). An off-target analysis of the mutants generated is shown. Twenty-nine SpCas 9-point mutants were generated and tested for specificity at (A) EMX1 target sites and (B) two VEGFA target sites. Mutants combined with superior residues with improved specificity were further tested in (C) EMX1 and (D) VEGFA. Annotated SpCas9 amino acid sequences are provided. Mutations in SpCas9 that altered the charge of the non-target strand groove were predominantly in the RuvC and HNH domains (highlighted in yellow). The RuvC (cyan), cross-linked helix (BH, green), REC (gray), HNH (magenta), and PI (beige) domains are annotated as in Nishimasu et al. A comparison of the specificity of K855A, eSpCas9 (1.0), and eSpCas9 (1.1) with truncated sgRNA is shown, and SpCas9 (1.0) and eSpCas9 (1.1) are used as strategies to improve the specificity. It shows that it is superior to the truncated sgRNA. Indel frequencies at three loci (EMX1 (1), VEGFA (1) and VEGFA (5)) were tested at major annotated predicted off-target sites. For both VEGFA target sites, tru-sgRNA increased the frequency of indels at some off-target sites, resulting in indels at off-target sites not found in the wild type. The number of off-target sites detectable by NGS for each SpCas9 mutant is listed below the heatmap. It is shown that increasing the positive charge of the nt groove can increase the cleavage at the off-target site. The point mutants SpCas9 (S845K) and SpCas9 (L847R) exhibited lower specificity at the EMX1 (1) target site than wild-type SpCas9. The production of eSaCas9 by mutagenesis of the nt groove is shown. An improved version of SaCas9 was produced similar to eSpCas9. (A, B) Single and double amino acid mutants of residues in the groove between the RuvC and HNH domains were screened for reduced off-target cleavage. (C) Mutants with improved specificity were combined to create mutants of SaCas9 in which on-target cleavage was maintained at EMX site 7 and off-target cleavage was significantly reduced. (D) Crystal structure of SaCas9 showing a groove between the HNH domain and the RuvC domain. We show the characterization of on-target efficiency for specific mutants that enhance specificity. Three SpCas9 mutants in the phosphate lock loop of the PI domain (Lys1107, Glu1108, Ser1109) confer specificity on bases 1 and 2 of the sgRNA proximal to the PAM. They consisted of a point mutant (K1107A) and two mutants in which the Lys-Glu-Ser sequence was replaced with the dipeptides Lys-Gly (KG) and Gly-Gly (GG), respectively. Our data have shown that these mutants can substantially reduce on-target cleavage efficiency. It shows that eSpCas9 (1.1) is not cytotoxic to human cells. HEK293T cells were transfected with WT or eSpCas9 (1.1), incubated for 72 hours, and then cell viability was measured using the CellTiter-Glo assay, which fluoresces in response to ATP production by living cells. Analysis of Nt groove mutants with truncated guide RNA is shown. Truncate-type guide RNA (Tru) was combined with a single amino acid SpCas9 mutant and targeted to (A) EMX1 (1) or (B) VEGFA (1). While many mutants targeting EMX1 with the 18 nt guide retained on-target efficiency, those targeting VEGFA (1) with the 17 nt guide were severely compromised. The single and double amino acid mutants selected are shown. As in SpCas9, the specificity increases as the positive charge of the non-targeting strand groove decreases. The reduction of the positive charge can be achieved by substituting the positively charged amino acids for neutral or negatively charged amino acids (A) or by moving the position of the positively charged amino acids in the groove (B). The mutant of interest is K572. It shows an improvement in the specificity of the selected mutant. CM2 exhibits a strong reduction in off-target activity while retaining complete on-target activity. CM1: R499A; Q500K; K572A. CM2: R499A; Q500K; R654A; G655R. CM3: K572A; R654A; G655R. It shows activation of the γ-globin HBG1 locus by a complex of spCas9 or spCas9 mutant guides of lengths 15 bp, 17 bp, and 20 bp. Cas9 (px165) is a non-mutated spCas9. dCas9 represents an inactive spCas9. The single mutants shown (“SM”) are R780A, K810A, and K848A. The double mutants shown (“DM”) are R780A / K810A and R780A / K855A. A comparison of various programmable nuclease platforms is shown. Indicates the type of therapeutic genomic modification. The specific type of genome editing therapy depends on the nature of the mutation that causes the disease. a. In gene silencing, targeting loci with NHEJ silences the pathogenic function of proteins. The formation of indels in the gene of interest often results in frameshift mutations to produce immature stop codons and non-functional protein products, or nonsense-mediated degradation of transcripts resulting in genes. Function is suppressed. b, HDR gene modification can be used to correct harmful mutations. DSBs are targeted in the vicinity of the mutation site in the presence of an extrinsically provided modified HDR template. HDR repair of this cleavage site with an exogenous template corrects the mutation and restores gene function. c. An alternative method for gene modification is gene addition. This treatment involves introducing a therapeutic transgene into a safe harbor locus in the genome. The DSB is targeted at the safe harbor locus and the HDR template, promoter and transgene, including homology to the cleavage site, are introduced into the nucleus. HDR repair copies the promoter-trans gene cassette to the Safe Harbor locus and restores gene function, but without true physiological control over gene expression. Ex vivo vs. in vivo editing therapy is shown. In ex vivo editing therapy, cells are removed from the patient, edited, and then retransplanted (top panel). For this treatment to be successful, the target cells must have the ability to survive in vitro and return to the target tissue after transplantation. In vivo therapy involves genome editing of cells in situ (bottom panel). For in vivo systemic therapies, the use of delivery agents that are relatively independent of cell identity or status can result in a wide range of histological edits. Although this editorial treatment method may be possible in the future, there is currently no efficient delivery system sufficient to make it feasible. In vivo targeted therapies, in which a delivery agent directed to a particular organ system is administered to a patient, are feasible using clinically relevant viral vectors. The schematic diagram of the gene therapy by Cas9 homologous recombination (HR) vector is shown. A schematic representation of glycosylation for protein or guided delivery, especially by GalNac, is provided. In addition, the sequence alignment of SaCas9 and SpCas9 is shown. The RUVC and HNH domain annotations for these two proteins are also shown in these three figures.

The figures herein are for illustrative purposes only and are not always drawn on a fixed scale.

Prior to describing the methods of the invention, the invention is not limited to the particular methods, components, products or combinations described, as such methods, components, products and combinations may of course differ. It should be understood that there is. It should also be understood that the terminology used herein is not intended to be limiting, as the scope of the invention is limited only by the appended claims. ..

As used herein, the singular forms "a," "an," and "the" include both the singular and the plural referents, unless expressly specified in the context.

The terms "comprising," "comprises," and "comprised of," as used herein, are "comprising." , "Includes" or "contining", "consisting", which are inclusive or non-restrictive and additional Do not exclude members, elements or method steps not listed. The terms "comprising," "comprises," and "comprised of," as used herein, are the terms "consisting." "of", "consists" and "consists of", and the terms "consisting essentially of", "consentially" and It will be understood to include "consentially of". In this disclosure and in particular the claims and / or paragraphs, terms such as "comprises," "complied," and "comprising" are US patents. They can have implications in the law; for example, they can mean "includes," "included," "includes," and so on. That; and terms such as "consisting essentially of" and "consistentally of" have meanings attributed to them in US patent law. For example, it is noted that these allow elements not explicitly described, but exclude elements found in the prior art or elements that affect the basic or novel features of the invention. In practicing the present invention, it may be advantageous to comply with Article 53 (c) EPC and Rule 28 (b) and (c) EPC. Nothing in this specification is intended to be promise.

The description of the numerical range by endpoint includes any number and fraction contained within each range, as well as the endpoints described.

The term "about" or "approximately" as used herein refers to, and ± 20% of the value indicated when referring to measurable values such as parameters, quantities, time periods, etc. Hereinafter, it is meant to include variations of preferably ± 10% or less, more preferably ± 5% or less, and even more preferably ± 1% or less, as long as such variations are appropriate for the practice of the disclosed invention. .. It should be understood that the values referenced by the modifier "about" or "approximately" are also specifically and preferably disclosed in their own right.

The term "one or more" or "at least one", eg, one or more or at least one member of a group of members, is self-evident, but as a further example, the term particularly said. References to any one of the members, or any two or more of the members, such as any of the members ≧ 3, ≧ 4, ≧ 5, ≧ 6 or ≧ 7, and up to all of the members. Is included.

All references cited herein are hereby incorporated by reference in their entirety. In particular, the teachings of all references specifically referenced herein are incorporated by reference.

Unless otherwise defined, all terms used in the disclosure of the present invention, including scientific and technological terms, have the meanings generally understood by those skilled in the art to which the present invention belongs. As a further guide, definitions of terms are included to better understand the teachings of the present invention.

In the following sections, various aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect, unless explicitly stated to be contrary to it. In particular, any feature indicated as preferred or advantageous can be combined with any other feature indicated as preferred or advantageous.

A standard reference describing the general principles of recombinant DNA technology is Molecular Cloning: A Laboratory Manual, 2nd ed. , Vol. 1-3, ed. Sambrook et al. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. et al. Y. , 1989; Current Protocols in Molecular Biology, ed. Ausubel et al. , Greene Publicing and Willy-Interscience, New York, 1992 (with periodic updates) ("Ausube et al. 1992"); the series Mechanism; Incorporated, Inc. , PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; PCR 2: A Practical Aproch (M.J. MacPherson, B.D.H.R.H. And Lane, eds. (1988) Antibodies, a Laboratory Manual; and Animal Cell Culture (RI Freshney, ed. (1987). General principles of microbial science include, for example, Davis, BD. et al., Microbiology, 3rd edition, Harper & Row, publics, Philadelphia, Pa. (1980).

References to "one embodiment" or "an embodiment" throughout the specification include detailed features, structures or properties described in connection with that embodiment in at least one embodiment of the invention. Means that. Thus, the phrase "in one embodiment" or "in one embodiment" appears in various places throughout the specification, but not all refer to the same embodiment, but it does. It can happen. Moreover, as will be apparent to those skilled in the art, such detailed features, structures or properties can be combined in any suitable manner in one or more embodiments. Moreover, some embodiments described herein include certain of the many features included in other embodiments, but different embodiments, as those skilled in the art will understand. A combination of morphological features is within the scope of the present invention and is intended to form different embodiments. For example, in the appended claims, any patented embodiment may be used in any combination.

In this description of the present invention, the accompanying drawings which form a part of the present specification are referred to, and the drawings are merely shown as examples of specific embodiments in which the present invention can be carried out. It should be understood that other embodiments may be utilized and structural or logical changes can be made without departing from the scope of the invention. Therefore, this description should not be construed in a limited sense and the scope of the invention is defined by the appended claims.

An object of the present invention is any previously known product for which Applicants reserve the right and disclose any previously known disclaimer of the product, process, or method herein. , The manufacturing process of the product, or the method of using the product is also not included in the scope of the present invention. In addition, the invention is intended to disclose by this specification any previously described product, product fabrication process, or discrimer of how the product is used, with which the Applicants reserve the right. Any product or product that does not meet the description and enablement requirements of the United States Patent and Trademark Office (USPTO) (US Patent Law, Article 112, paragraph 1) or the European Patent Office (EPO) (EPC, Article 83). It is noted that the process or method of use of the product is also intended not to be included within the scope of the invention.

Preferred description items (features) and embodiments of the present invention are shown below. Each of the statements and embodiments of the invention so defined may be combined with any other statement and / or embodiment unless explicitly stated to be contrary to it. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or description indicated as preferred or advantageous.

As used herein, the term "non-human organism" or "non-human cell" refers to an organism or cell that differs from or is not derived from human (Homo sapiens). As used herein, the term "non-human eukaryote" or "non-human eukaryote" refers to a eukaryote or cell that differs from or is not derived from human (Homo sapiens). In a preferred embodiment, such eukaryotes (cells) are non-human animals (cells) such as non-human mammals, non-human primates, ungulates, rodents (preferably mice or rats), rabbits, canine animals. , Dogs, cows, cows, sheep, sheep animals, goats, pigs, birds and birds, poultry, chickens, fish, insects, or arthropods, preferably mammals such as rodents, especially mice (cells or cells) Group). In some embodiments of the invention, the organism or subject or cell may be an arthropod, such as an insect, or a nematode (cell or cell population derived from). In some methods of the invention, the organism or subject or cell is a plant (cell). In some methods of the invention, the organism or subject or cell is an alga, including microalgae, or a fungus (derived from it). Those skilled in the art will prefer that eukaryotic cells that can be transplanted or introduced into non-human eukaryotes by the methods referred to herein are derived from the same species as the eukaryote into which they are transplanted. You will understand that it comes from it. For example, in certain embodiments, mouse cells are transplanted into mice by the methods of the invention as described herein. In certain embodiments, the eukaryote is an immunodeficient eukaryote, i.e., a eukaryote whose immune system is partially or completely arrested. For example, immunodeficient mice can be used in the methods according to the invention as described herein. Examples of immunodeficient mice include, but are not limited to, nude mice, RAG − / − mice, SCID (severe combined immunodeficiency) mice, SCID beige mice, NOD (non-obese diabetic) SCID mice, NOG or NSG mice. And so on.

It will be appreciated that the CRISPR-Cas system as described herein is not naturally present in the cells, i.e. engineered, or is exogenous to the cells. The CRISPR-Cas system as referred to herein has been introduced into the cells. Methods of introducing the CRISPR-Cas system into cells are known in the art and will be further described elsewhere herein. Cells containing the CRISPR-Cas system according to the present invention or into which the CRISPR-Cas system has been introduced establish a functional CRISPR complex capable of modifying (such as cleaving) a target DNA sequence. Contains or has the ability to express the individual constituents of the system. Thus, as referred to herein, cells containing the CRISPR-Cas system are individuals of the CRISPR-Cas system for establishing a functional CRISPR complex capable of modifying the target DNA sequence (such as cleaving). Can be a cell containing the constituents of. Alternatively, as referred to herein, and preferably, the cell containing the CRISPR-Cas system may be a cell containing one or more nucleic acid molecules encoding the individual components of the CRISPR-Cas system. , This nucleic acid molecule can be expressed in cells to establish a functional CRISPR complex capable of modifying the target DNA sequence (such as cleaving).

As used herein, the V-type or VI-type CRISPR-Cas locus effector protein is referred to as "crRNA" or "guide RNA" or "single guide RNA" or "sgRNA" or "one or more nucleic acid components". The term includes any polynucleotide sequence that has sufficient complementarity with the target nucleic acid sequence to hybridize with the target nucleic acid sequence and derive sequence-specific binding of the nucleic acid targeting complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97. When optimally aligned using a suitable alignment algorithm. 5%, 99% or more, or higher. Optimal alignment can be determined using any algorithm suitable for sequence alignment, examples of which are non-limiting examples: Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burroughs-Wheeler transformation based algorithm. (For example, Burroughs Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (available at Novocraft Technologies; www.novocaft.com), ELAND (Illumina, San Digigo, CA), SOC. (Available in) and Maq (Available in maq.Sourceforge.net). The ability of a guide sequence (within a nucleic acid targeting guide RNA) to induce sequence-specific binding of a nucleic acid targeting complex to a target nucleic acid sequence can be assessed by any suitable assay. For example, a component of the nucleic acid targeting CRISPR system sufficient to form a nucleic acid targeting complex is a component of the nucleic acid targeting complex into a host cell having the corresponding target nucleic acid sequence, including the guide sequence to be tested. It may be provided, for example, by transfection of a vector encoding the above, followed by evaluation of preferential targeting (eg, cleavage) within the target nucleic acid sequence, such as by a Surveyor assay as described herein. .. Similarly, cleavage of the target nucleic acid sequence provides the target nucleic acid sequence, the components of the nucleic acid targeting complex including the guide sequence to be tested, and the control guide sequence different from the test guide sequence, and the test thereof. It can be determined in vitro by comparing the binding or cleavage rate in the target sequence between the reactions of the guide sequence and the control guide sequence. Other assays are possible and will be recalled to those of skill in the art. The guide sequence, and thus the nucleic acid targeting guide RNA, can be selected to target any target nucleic acid sequence. The target sequence may be DNA. The target sequence may be any RNA sequence. In some embodiments, the target sequence is messenger RNA (mRNA), premRNA, ribosome RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), nuclear small molecule. Select from the group consisting of RNA (snRNA), small nuclear RNA (snoRNA), double-stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmic RNA (scRNA). It may be a sequence in the RNA molecule to be produced. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNAs and lncRNAs. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or pre-mRNA molecule.

In some embodiments, the nucleic acid targeting guide RNA is selected so that the degree of secondary structure within the RNA targeting guide RNA is reduced. In some embodiments, about 75%, 50%, 40%, 30%, 25%, 20% of the nucleotides of a nucleic acid targeting guide RNA are involved in self-complementary base pairing when optimally folded. , 15%, 10%, 5%, 1% or less, or less. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of such an algorithm is mFold as described by Zuker and Steigler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is an online web server RNAfold that uses a centroid structure prediction algorithm developed at the Institute for Theoretical Chemistry at the University of Vienna (eg, AR Grubber et al.). , 2008, Cell 106 (1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62).

In certain embodiments, the guide RNA or crRNA may include a direct repeat (DR) sequence and a guide sequence or spacer sequence, and may or may be essential. In certain embodiments, the guide RNA or crRNA may comprise, may be, or consist of, a direct repeat sequence fused or linked to a guide or spacer sequence. In certain embodiments, the direct repeat sequence may be located upstream (ie, 5'side) of the guide or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (ie, 3'side) of the guide or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably a single stem loop. In certain embodiments, the direct repeat sequence forms a stem-loop, preferably a single stem-loop.

In certain embodiments, the spacer length of the guide RNA is 15-35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is 15-17 nt, eg 15, 16, or 17 nt, 17-20 nt, eg 17, 18, 19, or 20 nt, 20-24 nt, eg 20, 21, 22. , 23, or 24 nt, 23-25 nt, eg 23, 24, or 25 nt, 24-27 nt, eg 24, 25, 26, or 27 nt, 27-30 nt, eg 27, 28, 29, or 30 nt, 30 ~ 35nt, for example 30, 31, 32, 33, 34, or 35nt, or 35nt or more.

The "tracrRNA" sequence or similar term includes any polynucleotide sequence that has sufficient complementarity to hybridize with the crRNA sequence. In some embodiments, the degree of complementarity between the tracrRNA sequence and the shorter length of the crRNA sequence when optimally aligned is about 25%, about 30%, about 40%. , About 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97.5%, about 99%, or higher, or more than about 25%, about 30 More than%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 95%, about 97.5 More than%, more than about 99%, or higher. In some embodiments, the tracr sequence is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 17, About 18, about 19, about 20, about 25, about 30, about 40, about 50, or more, or more than about 5, more than about 6, more than about 7, more than about 7, more than about 8, about 9. Exceeds, more than about 10, more than about 11, more than about 12, more than about 13, more than about 14, more than about 15, more than about 16, more than about 16, more than about 17, more than about 18, about 19 Nucleotide lengths greater than, greater than about 20, greater than about 25, greater than about 30, greater than about 40, greater than about 50, or greater. In some embodiments, the tracr and crRNA sequences are included within a single transcript such that hybridization between these sequences forms a transcript with a secondary structure, eg, a hairpin. In one embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In a preferred embodiment, the transcript has two, three, four, or five hairpins. In a further embodiment of the invention, the transcript has a maximum of 5 hairpins. In the hairpin structure, a portion of the sequence on the 5'side of the last "N" and upstream of the loop corresponds to the tracr mate sequence, and a portion of the sequence on the 3'side of the loop corresponds to the tracr sequence.

In general, the CRISPR-Cas, CRISPR-Cas9 or CRISPR system may be as used in the aforementioned documents such as International Publication No. 2014/093622 (PCT / US2013 / 074667), collectively. Cas gene, especially the sequence encoding the Cas9 gene in the case of CRISPR-Cas9, tracr (trans-activated CRISPR) sequence (eg tracrRNA or active partial CRISPRRNA), tracr mate sequence ("direct repeat" in the context of endogenous CRISPR systems And partial direct repeats processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or "RNA" (as the term is used herein). For example, CRISPR-related (including CRISPR RNA and CRISPR RNA and trans-activated (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from the CRISPR locus, such as those that guide Cas9. “Cas”) Refers to transcripts and other elements involved in or directing the expression of a gene. In general, the CRISPR system is characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence (also referred to as the protospacer in the context of the endogenous CRISPR system). In the context of the formation of a CRISPR complex, "target sequence" refers to a sequence in which the guide sequence is designed to be complementary to it, where hybridization between the target sequence and the guide sequence is CRISPR. Promotes the formation of complexes. The section of the guide sequence that provides complementarity to the target sequence important for cleavage activity is referred to herein as the seed sequence. The target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell and may include nucleic acids that are in or derived from mitochondria, organelles, vesicles, liposomes or intracellular particles. In some embodiments, NLS is not preferred, especially for non-nuclear applications. In some embodiments, direct repeats may be identified in silico by searching for repeating motifs that meet some or all of the following criteria: 1. It is present in the genomic sequence of a 2 Kb window adjacent to the type II CRISPR locus; spans 2.20-50 bp; and is only 3.20-50 bp apart. In some embodiments, two of these criteria, such as 1 and 2, 2 and 3, or 1 and 3, may be used. In some embodiments, all three criteria may be used.

In embodiments of the present invention, the term guide sequences and guide RNAs, ie RNAs capable of guiding Cas to target genomic loci, are described in WO 2014/093622 (PCT / US2013 / 074667) and the like. Used synonymously, as described in the aforementioned cited references. In general, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and induce sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is approximately 50%, 60%, 75%, 80% when optimally aligned using a suitable alignment algorithm. , 85%, 90%, 95%, 97.5%, 99% or more, or higher. Optimal alignment can be determined using any algorithm suitable for sequence alignment, examples of which are non-limiting examples: Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burroughs-Wheeler transformation based algorithm. (For example, Burroughs Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (available at Novocraft Technologies; www.novocraft.com), ELAND (Illumina, San Digigo, CA), SOAP. (Available) and Maq (available at maq.Sourceforge.net). In some embodiments, the guide sequences are approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer, or longer. In some embodiments, the guide sequence is about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length or less, or shorter. Preferably the guide sequence is 1030 nucleotides in length. The ability of the guide sequence to induce sequence-specific binding of the CRISPR complex to the target sequence can be assessed by any suitable assay. For example, transfection of a vector encoding a component of a CRISPR sequence into a host cell that has a corresponding target sequence, including a guide sequence to be tested, with sufficient components of the CRISPR system to form the CRISPR complex. It may be provided, such as by transfection, and can subsequently be assessed for preferential cleavage within the target sequence, such as by the Surveyor assay as described herein. Similarly, cleavage of the target polynucleotide sequence provides the target sequence, the components of the CRISPR complex, including the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and guides them to be tested. It can be determined in vitro by comparing the binding or cleavage rate in the target sequence between the reactions of the sequence and the control guide sequence. Other assays are possible and will be recalled to those of skill in the art.

In some embodiments of the CRISPR-Cas system, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95. %, 97.5%, 99%, or 100% or more; the guide or RNA or sgRNA is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer, or longer; or guide or RNA Alternatively, the sgRNA may be less than or shorter than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length; and advantageously the tracrRNA is 30 or 50 nucleotides in length. is there. However, one aspect of the invention is to reduce off-target interactions, eg, reduce guide interactions with less complementary target sequences. In fact, in these examples, between target and off-target sequences having 80% to greater than about 95% complementarity, eg, 83% to 84% or 88 to 89% or 94 to 95% complementarity. Mutations that result in a CRISPR-Cas system that can be distinguished (eg, distinguishing targets with 18 nucleotides from off-targets with 18 nucleotides with 1, 2 or 3 mismatches) are involved in the invention. Shown. Therefore, in the context of the present invention, the degree of complementarity between the guide sequence and its corresponding target sequence is 94.5% or 95% or 95.5% or 96% or 96.5% or 97%. Or 97.5% or 98% or 98.5% or 99% or 99.5% or more than 99.9%, or 100%. Off-targets are 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% between the sequence and the guide. Or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or Complementarity of less than 83% or 82% or 81% or 80%, advantageously off-target is 100% or 99.9% or 99.5% or 99% or 99 between its sequence and guide. % Or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity.

In a particularly preferred embodiment of the invention, the guide RNA (having the ability to guide Cas to the target locus) is (1) a guide sequence capable of hybridizing to the genomic target locus of eukaryotic cells; (2) tracr. Sequences; and (3) tracr mate sequences may be included. All (1) to (3) may be in a single RNA (arranged in the direction from 5'to 3'), i.e. in an sgRNA, or the tracr RNA is an RNA encoding a guide and tracr sequence. It may be an RNA different from. The tracr hybridizes to the tracr mate sequence and directs the CRISPR / Cas complex to the target sequence.

The methods according to the invention as described herein include eukaryotic cells as discussed herein (in vitro,) comprising delivering the vector as discussed herein to the cell. That is, it involves the step of inducing one or more mutations in (isolated eukaryotic cells). This one or more mutations include the introduction, deletion, or substitution of one or more nucleotides in each target sequence of one or more cells via one or more guide RNAs or sgRNAs. obtain. This mutation may include the introduction, deletion, or substitution of 1-75 nucleotides in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. This mutation includes 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides. This mutation includes 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides. This mutation includes 10,11,12,13,14,15,16,17,18,19, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides. This mutation includes 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 30, 35, 40, 45, 50, or 75 nucleotides. The mutation involves the introduction of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides in each target sequence of the cell via one or more guide RNAs or sgRNAs. , Deletion, or substitution can be included.

Control of the concentration of delivered Cas mRNA and guide RNA is considered to minimize toxicity and off-target effects. Optimal concentrations of Cas mRNA and guide RNA should be determined by testing various concentrations in cellular or non-human eukaryotic animal models and analyzing the degree of alteration at potential off-target genomic loci using deep sequencing. Can be done. Alternatively, in order to minimize the level of toxicity and off-target effects, Cas nickase mRNA (eg, S. pyogenes Cas9 with a D10A mutation) is used as a pair of guide RNAs that target the site of interest. Can be delivered with. Guide sequences and strategies for minimizing toxicity and off-target effects may be as described in WO 2014/093622 (PCT / US2013 / 074667); or herein. It may be mutated as described in.

Typically, in the context of an endogenous CRISPR system, once a CRISPR complex, including a guide sequence that hybridizes to a target sequence and forms a complex with one or more Cas proteins, is formed, the target sequence. One or both at or near the target sequence (eg, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs, or more) Breaking of the chain occurs. Although not desired to be constrained by theory, all or part of the wild-type tracr sequence (eg, about 20, 26, 32, 45, 48, 54, 63, 67, 85 nucleotides or more of the wild-type tracr sequence). The tracr sequence that can contain or consist of (or more) is also hybridized along at least a portion of the tracr sequence to all or part of the tracr mate sequence operably linked to the guide sequence. , Can form part of the CRISPR complex.

The locations of the RuvC, RuvCII, RuvCIII and HNH domains are shown in FIGS. 22A-22C. As used herein, the term "RuvCI domain" is preferably in a domain containing Amino acids 1-60 of Streptococcus pyogenes Cas9 (SpCas9) or in another Cas9 ortholog or a CRISPR nuclease other than Cas9. Refers to the corresponding area. As used herein, the term "RuvCII domain" is preferably used in a domain containing the amino acids 718-775 of Streptococcus pyogenes Cas9 (SpCas9) or in another Cas9 ortholog or a CRISPR nuclease other than Cas9. Refers to the corresponding area. As used herein, the term "RuvCIII domain" is preferably in a domain containing the amino acids 909-1099 of Streptococcus pyogenes Cas9 (SpCas9) or in another Cas9 ortholog or a CRISPR nuclease other than Cas9. Refers to the corresponding area. As used herein, the term "HNH domain" is preferably in a domain containing the amino acids 776-908 of Streptococcus pyogenes Cas9 (SpCas9) or in another Cas9 ortholog or a CRISPR nuclease other than Cas9. Refers to the corresponding area. The groove between the RuvC domain and the HNH domain refers to the groove between these domains in the three-dimensional structure of the non-naturally occurring CRISPR enzyme as described herein. FIG. 25D shows the crystal structure of SaCas9, showing the groove between the HNH domain and the RuvC domain in the three-dimensional structure of SaCas9.

Aptamer One guide with a first aptamer / RNA-binding protein pair can be linked or fused to an activator, while a second guide with a second aptamer / RNA-binding protein pair can be linked or fused to a repressor. Can be fused. The targets (loci) of these guides are different, so it is possible to activate one gene and suppress one gene. For example, the following schematic shows such a technique:
Guide 1-MS2 aptamer-MS2 RNA-binding protein-VP64 activator; and Guide 2-PP7 aptamer-PP7 RNA-binding protein-SID4x repressor.

The present invention also relates to orthogonal PP7 / MS2 gene targeting. In this example, MS2-VP64 or PP7-SID4X are recruited by modifying sgRNAs targeting different loci with individual RNA loops, which activate and suppress their respective target loci. PP7 is an RNA-binding coat protein of the genus Pseudomonas of the genus Bacteriophage Pseudomonas. Like MS2, it binds to specific RNA sequences and secondary structures. The PP7 RNA recognition motif is different from that of MS2. As a result, PP7 and MS2 can be multiplexed so that individual effects are mediated simultaneously at different genomic loci. For example, an sgRNA targeting locus A can be modified in the MS2 loop to recruit the MS2-VP64 activator, while another sgRNA targeting locus B can be modified in the PP7 loop to PP7-. The SID4X repressor domain can be recruited. Thus, in the same cell, dCas9 can mediate orthogonal locus-specific alterations. This principle can be extended to the uptake of other orthogonal RNA-binding proteins such as Q-β.

An alternative option for orthogonality suppression is to use a non-coding RNA loop with an interactive suppression function as a guide (either at the same position as the MS2 / PP7 loop incorporated in the guide, or at the 3'end of the guide. Crab) can be taken in. For example, guides with non-coding (but known to be inhibitory) RNA loops were designed (eg, using an Alu repressor (which is RNA) that interferes with RNA polymerase II in mammalian cells). This Alu RNA sequence was in place of the MS2 RNA sequence as used herein (eg, in tetraloop and / or stemloop 2); and / or was located at the 3'end of the guide. This may result in a combination of MS2, PP7 or Alu at the tetraloop and / or stemloop 2 positions, and optionally adds Alu (with or without a linker) to the 3'end of the guide. It will be.

With two different aptamers (individual RNAs), activator-adapter protein fusions and repressor-adapter protein fusions are used with different guides to activate the expression of one gene while another. It becomes possible to suppress the expression of. These can be administered together or substantially together in a multiplexing technique, along with their different guides. Since a relatively small number of Cas9s can be used with a large number of modified guides, only one (or at least a minimum number) while using a large number of such modified guides all at the same time, for example 10 or 20 or 30 All you have to do is deliver Cas9. The adapter protein may be associated (preferably linked or fused) with one or more activators or one or more repressors. For example, the adapter protein may be associated with a first activator and a second activator. The first and second activators may be the same, but they are preferably different activators. For example, one may be VP64 and the other may be p65, but these are just examples and other transcription activators are envisioned. Three or more or even four or more activators (or repressors) may be used, but package size may limit the number of different functional domains to more than five. Compared to direct fusion with the adapter protein, a linker is preferably used, where two or more functional domains associate with the adapter protein. Suitable linkers include GlySer linkers.

It is also envisioned that the enzyme-guide complex as a whole may associate with more than one functional domain. For example, there may be more than one functional domain associated with the enzyme, or there may be more than one functional domain associated with the guide (via one or more adapter proteins), or associated with the enzyme. There may be one or more functional domains and one or more functional domains associated with the guide (via one or more adapter proteins).

The fusion between the adapter protein and the activator or repressor may include a linker. For example, the GlySer linker GGGS can be used. By using these in 3 ((GGGGS) ₃ ) or 6, 9 or even 12 or more iterations, suitable lengths can be provided as needed. Linkers can be used between RNA-binding proteins and functional domains (activators or repressors), or between CRISPR enzymes (Cas9) and functional domains (activators or repressors). Linker, the user manipulates the appropriate amount of "mechanical flexibility".

Dead Guide: In one aspect in which a guide RNA containing a dead guide sequence can be used in the present invention, the present invention allows successful formation of a CRISPR complex and binding to a target while not allowing successful nuclease activity ( That is, it provides a guide sequence that is modified in the form of no nuclease activity / no indel activity). For explanatory purposes, such modified guide sequences are referred to as "dead guides" or "dead guide sequences." These dead guides or dead guide sequences can be considered catalytically inactive or sterically inactive with respect to nuclease activity. The nuclease activity can be measured using surveyor analysis or deep sequencing, preferably using surveyor analysis, as is commonly used in the art. Similarly, dead guide sequences may not be fully involved in productive base pairing with respect to their ability to promote catalytic activity or distinguish between on-target and off-target binding activities. Briefly, the surveyor assay involves purification and amplification of the CRISPR target site of a gene and formation of heteroduplexes with primers that amplify the CRISPR target site. After annealing, the product is treated with SURVEYOR nuclease and SURVEYOR Enhancer S (Transgenomics) according to the manufacturer's recommended protocol, analyzed on gel and quantified based on relative band strength.

Thus, in a related aspect, the invention provides a non-naturally occurring or engineered composition Cas9 CRISPR-Cas system comprising functional Cas9 as described herein, and a guide RNA (gRNA). Here, the gRNA contains a dead guide sequence, whereby the gRNA has a Cas9 CRISPR-Cas system without detectable indel activity resulting from the nuclease activity of the non-mutated Cas9 enzyme of this system as detected by the SURVEYOR assay. It will have the ability to hybridize to a target sequence that leads to the target genomic locus in the cell. For brevity, a Cas9 CRISPR-Cas system that contains a dead guide sequence and thus without detectable indel activity resulting from the nuclease activity of the non-mutated Cas9 enzyme of this system as detected by the SURVEYOR assay. A gRNA having the ability to mutate to a target sequence such that the gRNA leads to the genomic locus of interest in the cell is referred to herein as a "dead gRNA". It is understood that any of the gRNAs according to the invention as described elsewhere herein can be used as gRNAs containing dead gRNA / dead guide sequences as described below herein. Should be. Any of the methods, products, compositions and uses as described elsewhere herein are equally applicable with gRNAs containing dead gRNA / dead guide sequences as further detailed below. As further guidance, the following detailed embodiments and embodiments are provided.

The ability of the dead guide sequence to guide sequence-specific binding of the CRISPR complex to the target sequence can be assessed by any suitable assay. For example, a vector encoding a CRISPR sequence component to a host cell that has a corresponding target sequence, including a dead guide sequence to be tested, that has sufficient components of the CRISPR system to form the CRISPR complex. It may be provided, for example by transfection, and subsequently preferred cleavage within the target sequence can be assessed, such as by the Surveyor assay as described herein. Similarly, cleavage of the target polynucleotide sequence provides the target sequence, the components of the CRISPR complex, including the dead guide sequence to be tested, and a control guide sequence different from the test dead guide sequence. It can be determined in vitro by comparing the binding or cleavage rate in the target sequence between the reactions of the test guide sequence and the control guide sequence. Other assays are possible and will be recalled to those of skill in the art. The dead guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell.

As further described herein, some structural parameters allow the appropriate framework to lead to such dead guides. Dead guide sequences are shorter than their respective guide sequences, resulting in active Cas9-specific indel formation. Dead guides are 5%, 10%, 20%, 30%, 40%, 50% shorter than their respective guides targeting the same Cas9, resulting in active Cas9-specific indel formation.

As described below and known in the art, one aspect of gRNA-Cas9 specificity is a direct repeat sequence, which should be properly linked to such a guide. In particular, this implies that the direct repeat sequence is designed according to the origin of Cas9. Therefore, structural data available for verified dead guide sequences can be used in the design of Cas9 specific equivalents. For example, structural similarities between the orthologous nuclease domain RuvC of two or more Cas9 effector proteins can be used to transfer a dead guide of equivalent design. Thus, the dead guides herein are appropriately modified in length and sequence to reflect such Cas9-specific equivalents, thereby achieving successful CRISPR complex formation and binding to the target, as well as at the same time. Successful nuclease activity becomes unacceptable.

The use of dead guides in the context of this specification and the state of the art in the art allows for multiple gene targeting, in particular bidirectional multiple gene targeting, both in vitro, ex vivo, and in vivo applications in network biology and /. Or it brings a surprising and unexpected platform for systems biology. Prior to the use of dead guides, addressing multiple targets, for example for activation, suppression and / or silencing of gene activity, was a challenge and in some cases impossible. Dead guides can be used, for example, to address multiple targets, and thus multiple activities, in the same cell, the same animal, or the same patient. Such multiplexing may occur simultaneously or staggered at a desired time frame.

For example, here the dead guide, for the first time, allows the use of gRNA as a means of gene targeting without nuclease activity, while at the same time providing a guided activation or suppression means. Guide RNAs, including dead guides, allow the functional placement of elements, specifically gene effectors (eg, gene activity activators or repressors), in a manner that allows activation or suppression of gene activity. It can be modified to further include a protein adapter (eg, an aptamer) as described elsewhere in the specification. One example is the incorporation of aptamers as described herein and at the technical level of the art. By engineering the gRNA containing the dead guide to incorporate protein-interactive aptamers (Konermann et al., "Genome-scale transcription activation by genome-scale transcription activation by engineered CRISPR-Cas9 complex". CRISPR-Cas9 protein) ”, doi: 10.1038 / genome14136, incorporated herein by reference), synthetic transcriptional activation complexes consisting of multiple individual effector domains can be assembled. This can be modeled after the natural transcriptional activation process. For example, an aptamer that selectively binds to an effector (eg, an activator or repressor; a dimerized MS2 bacteriophage coat protein as a fusion protein with an activator or repressor), or an effector itself (eg, an activator). Or a protein that binds to the repressor) can be added to the dead gRNA tetraloop and / or stem-loop 2. In the case of MS2, the fusion protein MS2-VP64 binds to tetraloop and / or stem-loop 2 and then mediates up-transcriptional regulation of, for example, Neurog2. Other transcriptional activators are, for example, VP64, P65, HSF1, and MyoD1. As an example of this concept, the suppressor element can be recruited by replacing the MS2 stem-loop with a PP7 interaction stem-loop.

Accordingly, one aspect is a gRNA of the invention comprising a dead guide, which gRNA further comprises modifications that result in gene activation or suppression, as described herein. A dead gRNA can contain one or more aptamers. Aptamers may be specific for gene effectors, gene activators or gene repressors. Alternatively, the aptamer may be protein-specific, which in turn is specific for a particular gene effector, gene activator or gene repressor and recruits / binds to it. If there are multiple recruiting sites for the activator or repressor, it is preferred that those sites are specific to either the activator or the repressor. If there are multiple activator or repressor binding sites, those sites may be specific for the same activator or repressor. Those sites may also be specific for different activators or different repressors. Gene effectors, gene activators, and gene repressors can exist in the form of fusion proteins.

In certain embodiments, the dead gRNA as described herein or the Cas9 CRISPR-Cas complex as described herein is a non-naturally occurring or engineered containing two or more adapter proteins. Contains the composition, where each protein associates with one or more functional domains, and the adapter protein is linked to one or more individual RNA sequences inserted into at least one loop of dead gRNA. ..

Thus, in one embodiment, a guide RNA (gRNA) comprising a dead guide sequence capable of hybridizing to a target sequence at the genomic locus of interest in the cell (where the dead guide sequence is as defined herein). Cas9 comprising at least one or more nuclear localization sequences, optionally providing a non-naturally occurring or engineered composition comprising Cas9 comprising at least one mutation, wherein of the dead gRNA. At least one loop is modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and is the adapter protein associated with one or more functional domains; or dead gRNAs Modified to have at least one non-coding functional loop, and the composition comprises two or more adapter proteins, each protein associated with one or more functional domains.

In certain embodiments, the adapter protein is a fusion protein comprising a functional domain, the fusion protein optionally comprises a linker between the adapter protein and the functional domain, and the linker optionally comprises a GlySer linker.

In certain embodiments, at least one loop of dead gRNA is not modified by the insertion of one or more individual RNA sequences that bind to two or more adapter proteins.

In certain embodiments, the one or more functional domains associated with the adapter protein are transcriptional activation domains.

In certain embodiments, one or more functional domains associated with the adapter protein are transcriptional activation domains, including VP64, p65, MyoD1, HSF1, RTA or SET7 / 9.

In certain embodiments, the one or more functional domains associated with the adapter protein are transcriptional repressor domains.

In certain embodiments, the transcriptional repressor domain is the KRAB domain.

In certain embodiments, the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or SID4X domain.

In certain embodiments, at least one of the one or more functional domains associated with the adapter protein is methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, DNA integration activity. Has one or more activities including RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity.

In certain embodiments, the DNA cleavage activity is due to Fok1 nuclease.

In certain embodiments, the dead gRNA binds to the adapter protein, and after further binding to Cas9 and the target, the dead gRNA is modified to be a functional domain in a spatial arrangement that allows the functional domain to function with its attribution function. ..

In certain embodiments, at least one loop of dead gRNA is a tetraloop and / or loop 2. In certain embodiments, the dead gRNA tetraloop and loop 2 are modified by the insertion of one or more individual RNA sequences.

In certain embodiments, the insertion of one or more individual RNA sequences that bind to one or more adapter proteins is an aptamer sequence. In certain embodiments, the aptamer sequence is two or more aptamer sequences specific for the same adapter protein. In certain embodiments, the aptamer sequence is two or more aptamer sequences specific for different adapter proteins.

In certain embodiments, the adapter proteins are MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, PRR1 are included.

In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells and optionally mouse cells. In certain embodiments, the mammalian cell is a human cell.

In certain embodiments, the first adapter protein associates with the p65 domain and the second adapter protein associates with the HSF1 domain.

In certain embodiments, the composition has Cas9 CRISPR- having at least three functional domains, at least one of which is associated with Cas9 and at least two of which are associated with dead gRNA. Includes Cas complex.

In certain embodiments, the composition further comprises a second gRNA, wherein the second gRNA is a live gRNA capable of hybridizing to a second target sequence, the second Cas9 CRISPR-Cas system. It is directed to a second genomic locus of interest in the cell, which is accompanied by detectable Indel activity at the second genomic locus resulting from the nuclease activity of the Cas9 enzyme in the system.

In certain embodiments, the composition further comprises a plurality of dead gRNAs and / or a plurality of live gRNAs.

One aspect of the present invention utilizes the modularity and customizability of a gRNA scaffold to construct a series of gRNA scaffolds with different binding sites (specifically aptamers) for orthogonally recruiting different types of effectors. Is. Again, as an example and explanation of the broader concept, the suppressor elements may be coupled / recruited by replacing the MS2 stem-loop with a PP7 interaction stem-loop and are multiplexed bidirectional. Transcription control may be possible. Therefore, in general, the use of gRNAs containing dead guides can provide multiple transcriptional regulation and preferred bidirectional transcriptional regulation. This transcriptional regulation is the most preferred of the gene. For example, one or more gRNAs containing one or more dead guides may be used to target activation of one or more target genes. At the same time, one or more gRNAs containing one or more dead guides can be used to target suppression of one or more target genes. Such sequences can be applied in a variety of different combinations, for example, first suppressing the target gene and then activating other targets at appropriate intervals, or the selected gene is the activity of the selected gene. It is suppressed at the same time as the conversion, followed by further activation and / or suppression. As a result, multiple components of one or more biological systems can be advantageously dealt with together.

In some embodiments, the invention provides one or more nucleic acid molecules encoding a dead gRNA or Cas9 CRISPR-Cas complex or composition as described herein.

In some embodiments, the invention provides a vector system comprising a nucleic acid molecule encoding a dead guide RNA as defined herein. In certain embodiments, the vector system further comprises one or more nucleic acid molecules encoding Cas9. In certain embodiments, the vector system further comprises one or more nucleic acid molecules encoding a (live) gRNA. In certain embodiments, the nucleic acid molecule or vector can act on a nucleic acid molecule encoding a guide sequence (gRNA) and / or a nucleic acid molecule encoding Cas9 and / or optionally one or more nuclear localization sequences. It further comprises one or more regulatory elements that can operate in eukaryotic cells linked to.

In another embodiment, structural analysis can also be used to study the interaction between the dead guide and the active Cas9 nuclease, which allows DNA binding but not DNA cleavage. In this way, amino acids important for Cas9 nuclease activity are determined. Modification of such amino acids results in the improved Cas9 enzyme used for gene editing.

A further aspect is to combine the use of dead guides as described herein with other applications of CRISPR as described herein and known in the art. For example, a gRNA containing one or more dead guides for targeted multiple gene activation or suppression or targeted multiple bidirectional gene activation / suppression, as described herein It may be combined with a gRNA containing a maintenance guide. Such gRNAs containing guides that maintain nuclease activity may or may not further contain modifications (eg, aptamers) that allow suppression of gene activity. Such gRNAs containing guides that maintain nuclease activity may or may not further contain modifications (eg, aptamers) that allow activation of gene activity. In this way, further means of multiple gene regulation are introduced (eg, multiple gene targeting activation without nuclease activity / indel activity at the same time as or in combination with gene targeting inhibition with nuclease activity). Can be drowned).

For example, 1) one or more gRNAs containing one or more dead guides targeting one or more genes and further modified with a suitable aptamer for recruiting gene activators (eg, 1-50, Use of 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5); 2) include one or more dead guides targeting one or more genes. And one or more gRNAs further modified with suitable activators for gene repressor recruitment (eg, 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1). ~ 5) may be combined. Then 1) and / or 2) are added to 3) one or more gRNAs targeting one or more genes (eg 1-50, 1-40, 1-30, 1-20, preferably 1-10. , More preferably 1 to 5). This combination is then also 1) + 2) + 3), 4) one or more gRNAs targeting one or more genes and further modified with a suitable aptamer for recruiting gene activators (eg 1). 50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) can be combined. This combination is then also 1) + 2) + 3) + 4), 5) one or more gRNAs targeting one or more genes and further modified with a suitable aptamer for recruiting gene repressors (eg, one or more gRNAs). 1 to 50, 1 to 40, 1 to 30, 1 to 20, preferably 1 to 10, more preferably 1 to 5) can be carried out together. As a result, various uses and combinations are included in the present invention. For example, combination 1) +2); combination 1) +3); combination 2) +3); combination 1) +2) +3); combination 1) +2) +3) +4); combination 1) +3) +4); combination 2) +3 ) +4); Combination 1) +2) +4); Combination 1) +2) +3) +4) +5); Combination 1) +3) +4) +5); Combination 2) +3) +4) +5); Combination 1) +2) +4 ) + 5); Combination 1) + 2) + 3) + 5); Combination 1) + 3) + 5); Combination 2) + 3) + 5); Combination 1) + 2) + 5).

In some embodiments, the present invention provides an algorithm for designing, evaluating, or selecting a dead guide RNA targeting sequence (dead guide sequence) for guiding the Cas9 CRISPR-Cas system to a target locus. In particular, it has been determined that the specificity of dead guide RNA is related to i) GC content and ii) targeting sequence length and can be optimized by altering it. In some embodiments, the invention provides an algorithm for designing or evaluating dead guide RNA targeting sequences that minimize off-target binding or interaction of dead guide RNA. In certain embodiments of the invention, an algorithm for selecting a dead guide RNA targeting sequence to guide a CRISPR system to an organism's locus a) locates one or more CRISPR motifs at the locus and i ) Analyze the downstream 20 nt sequence of each CRISPR motif by determining the GC content of the sequence; and ii) determining if the genome of the organism has an off-target match of the downstream 15 nucleotides closest to the CRISPR motif, and c) It involves selecting the 15 nucleotide sequence to use for the dead guide RNA when the GC content of the sequence is 70% or less and no off-target match is identified. In certain embodiments, when the GC content is 60% or less, the sequence is selected as the targeting sequence. In certain embodiments, when the GC content is 55% or less, 50% or less, 45% or less, 40% or less, 35% or less or 30% or less, the sequence is selected for the targeting sequence. In certain embodiments, two or more sequences of loci are analyzed and the sequence having the lowest GC content, or the next lowest GC content, or the next lowest GC content is selected. In certain embodiments, if no off-target match is identified in the genome of the organism, the sequence is selected for the targeting sequence. In certain embodiments, the targeting sequence is selected when no off-target match is identified in the regulatory sequence of the genome.

In some embodiments, the present invention provides a method of selecting a dead-guided RNA targeting sequence for directing a functionalized CRISPR system to an organism's locus, wherein a) one or more CRISPR motifs at the locus. Positioning steps; b) analyze the downstream 20 nt sequence of each CRISPR motif by i) determining the GC content of the sequence; and ii) determining if the genome of the organism has the first 15 nt off-target match of the sequence. Steps to: c) include selecting the sequence for use in a guide RNA if the GC content of the sequence is 70% or less and no off-target match is identified. In certain embodiments, the sequence is selected when the GC content is 50% or less. In certain embodiments, the sequence is selected when the GC content is 40% or less. In certain embodiments, the sequence is selected when the GC content is 30% or less. In certain embodiments, two or more sequences are analyzed and the sequence with the lowest GC content is selected. In certain embodiments, off-target matching is determined by the regulatory sequence of the organism. In certain embodiments, the locus is a regulatory region. One embodiment provides a dead guide RNA containing a targeting sequence selected by the method described above.

In some embodiments, the invention provides a dead guide RNA for targeting a functionalized CRISPR system to an organism locus. In certain embodiments of the invention, the dead guide RNA comprises a targeting sequence in which the CG content of the target sequence is 70% or less, the first 15 nt of this targeting sequence is CRISPR in the regulatory sequence of another locus of the organism. Does not match off-target sequences downstream from the motif. In certain embodiments, the GC content of the targeting sequence is 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less or 30% or less. In certain embodiments, the GC content of the targeting sequence is 70% -60% or 60% -50% or 50% -40% or 40% -30%. In certain embodiments, the targeting sequence has the lowest CG content of any potential targeting sequence at that locus.

In certain embodiments of the invention, the first 15 nt of the dead guide matches the target sequence. In another embodiment, the first 14 nt of the dead guide matches the target sequence. In another embodiment, the first 13 nt of the dead guide matches the target sequence. In another embodiment, the first 12 nt of the dead guide matches the target sequence. In another embodiment, the first 11nt of the dead guide matches the target sequence. In another embodiment, the first 10 nt of the dead guide matches the target sequence. In one embodiment of the invention, the first 15 nt of the dead guide does not match the off-target sequence downstream from the CRISPR motif in the regulatory region of another locus. In other embodiments, the first 14 nt of the dead guide, or the first 13 nt, or the first 12 nt of the guide, or the first 11 nt of the dead guide, or the first 10 nt of the dead guide is in the regulatory region of another locus. Does not match off-target sequences downstream from the CRISPR motif. In other embodiments, the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt of the dead guide does not match off-target sequences downstream from the CRISPR motif in the genome.

In certain embodiments, the dead guide RNA contains an additional nucleotide at the 3'end that does not match the target sequence. Thus, dead guide RNAs containing the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt downstream of the CRISPR motif are 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt at the 3'end. , Or the length may be extended to more than that.

The present invention provides a method of directing a Cas9 CRISPR-Cas system to a locus, including but not limited to a dead Cas9 (dCas9) or a functionalized Cas9 system, which may include a functionalized Cas9 or a functionalized guide. .. In some embodiments, the invention provides a method of selecting dead guide RNA targeting sequences and directing a functionalized CRISPR system to an organism locus. In some embodiments, the invention provides a method of selecting a dead guide RNA targeting sequence and causing gene regulation of a target locus by a functionalized Cas9 CRISPR-Cas system. In certain embodiments, the method is used to generate target gene regulation while minimizing off-target effects. In some embodiments, the present invention provides a method of selecting two or more dead guide RNA targeting sequences and causing gene regulation of two or more target loci by a functionalized Cas9 CRISPR-Cas system. In certain embodiments, the method is used to generate regulation of two or more target loci while minimizing off-target effects.

In certain embodiments, the present invention provides a method of selecting a dead guide RNA targeting sequence for directing functionalized Cas9 to an organism's locus, which method a) positions one or more CRISPR motifs at the locus. Steps to identify; b) the downstream sequence of each CRISPR motif is analyzed by i) selection of 10-15 nt adjacent to the CRISPR motif, ii) determination of the GC content of the sequence; and c) the GC content of the sequence In the case of 40% or more, the step of selecting the 10 to 15 nt sequence as the targeting sequence to be used for the guide RNA is included. In certain embodiments, the sequence is selected when the GC content is 50% or greater. In certain embodiments, the sequence is selected when the GC content is 60% or greater. In certain embodiments, the sequence is selected when the GC content is 70% or greater. In certain embodiments, two or more sequences are analyzed and the sequence with the highest GC content is selected. In certain embodiments, the method further comprises adding a nucleotide that does not match the sequence downstream of the CRISPR motif to the 3'end of the selected sequence. One embodiment provides a dead guide RNA containing a targeting sequence selected by the method described above.

In some embodiments, the invention provides a dead guide RNA for directing a functionalized CRISPR system to an organism's locus, where the targeting sequence of the dead guide RNA is 10 to 15 nucleotides flanking the CRISPR motif at the locus. The CG content of the target sequence is 50% or more. In certain embodiments, the dead guide RNA further comprises a nucleotide added to the 3'end of the targeting sequence that does not match the sequence downstream of the CRISPR motif at the locus.

In some embodiments, the invention provides a single effector that is directed to one or more or more than one locus. In certain embodiments, the effector associates with Cas9 and uses one or more, or two or more selected dead guide RNAs to allow the Cas9 associated effector to have one or more selected target genes. Guided to the seat. In certain embodiments, the effector associates with one or more selected dead guide RNAs, and when each selected dead guide RNA forms a complex with the Cas9 enzyme, the associated effector. Is localized to the dead guide RNA target. One non-limiting example of such a CRISPR system regulates the activity of one or more or more loci that are regulated by the same transcription factors.

In some embodiments, the invention provides two or more effectors that are directed to one or more loci. In certain embodiments, two or more dead guide RNAs are used, each of the two or more effectors is associated with a selected dead guide RNA, and each of the two or more effectors is of that dead guide RNA. Localize to the target of choice. One non-limiting example of such a CRISPR system regulates the activity of one or more loci that are regulated by different transcription factors. Thus, in one non-limiting embodiment, two or more transcription factors are localized to different regulatory sequences of a single gene. In another non-limiting embodiment, two or more transcription factors are localized to different regulatory sequences of different genes. In certain embodiments, one transcription factor is an activator. In certain embodiments, one transcription factor is an inhibitor. In certain embodiments, one transcription factor is an activator and the other transcription factor is an inhibitor. In certain embodiments, loci expressing different components of the same regulatory pathway are regulated. In certain embodiments, loci expressing components of different regulatory pathways are regulated.

In some embodiments, the invention also provides methods and algorithms for designing and selecting dead guide RNAs specific for target DNA cleavage or target binding and gene regulation mediated by the active Cas9 CRISPR-Cas system. In certain embodiments, the Cas9 CRISPR-Cas system provides orthogonal gene regulation with active Cas9 that cleaves the target DNA at one locus and at the same time binds to another locus and promotes its regulation.

In some embodiments, the invention provides a method of selecting a dead-guided RNA targeting sequence for directing functionalized Cas9 to an organism's locus without cleavage, wherein the method is a) one or more at the loci. Steps to locate CRISPR motifs; b) Analyze downstream sequences of each CRISPR motif by i) selection of 10-15 nt adjacent to CRISPR motifs, ii) determination of GC content of sequences, and c) sequences Further includes the step of selecting the 10 to 15 nt sequence as the targeting sequence to be used for the dead guide RNA when the GC content of the CRISPR is more than 30% and 40% or more. In certain embodiments, the GC content of the targeting sequence is 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, or 70% or higher. In certain embodiments, the GC content of the targeting sequence is 30% -40% or 40% -50% or 50% -60% or 60% -70%. In one embodiment of the invention, two or more sequences of loci are analyzed and the sequence with the highest GC content is selected.

In certain embodiments of the invention, the portion of the targeting sequence whose GC content is assessed is 10 to 15 contiguous nucleotides of the 15 target nucleotides closest to PAM. In certain embodiments of the invention, the portion of the guide in which the GC content is considered is 10-11 nucleotides or 11-12 nucleotides or 12-13 nucleotides or 13 or 14 of the 15 nucleotides closest to PAM, or It is 15 consecutive nucleotides.

In some embodiments, the invention further provides an algorithm for identifying dead guide RNAs that promote CRISPR locus cleavage while avoiding functional activation or inhibition. An increase in GC content of 16-20 nucleotides in the dead guide RNA is observed to be consistent with an increase in DNA cleavage and a decrease in functional activation.

It is also demonstrated herein that the efficiency of functionalized Cas9 can be increased by adding nucleotides that do not match the target sequence downstream of the CRISPR motif to the 3'end of the guide RNA. For example, of the 11-15 nt lengths of dead guide RNA, shorter guides may be less likely to promote target cleavage, but are also less efficient at promoting binding and functional control of the CRISPR system. In certain embodiments, the addition of nucleotides that do not match the target sequence to the 3'end of the dead guide RNA increases activation efficiency without increasing unwanted target cleavage. In some embodiments, the invention also provides methods and algorithms for identifying improved dead guide RNAs that effectively promote the function of the CRISPRP system in DNA binding and gene regulation but do not promote DNA cleavage. Thus, in certain embodiments, the invention comprises the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt downstream of the CRISPR motif, and 12 nt, 13 nt, 14 nt, 15 nt, depending on the nucleotide that is mismatched with the target. It provides a dead guide RNA with an extended 3'end length of 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, or more.

In some embodiments, the present invention provides a method of producing selective orthogonal gene regulation. As will be appreciated from the disclosure herein, dead guide selection according to the invention considering guide length and GC content results in effective and selective transcriptional regulation by the functional Cas9 CRISPR-Cas system. For example, activation or inhibition regulates locus transcription and minimizes off-target effects. Accordingly, the present invention also provides effective orthogonal regulation of two or more target loci by providing effective regulation of individual target loci.

In certain embodiments, orthogonal gene regulation is by activation or inhibition of two or more target loci. In certain embodiments, orthogonal gene regulation is by activation or inhibition of one or more target loci and cleavage of one or more target loci.

In one aspect, the invention provides a cell comprising a non-naturally occurring Cas9 CRISPR-Cas system comprising one or more dead guide RNAs disclosed or produced by the methods or algorithms described herein. In, the expression of one or more gene products is altered. In certain embodiments of the invention, the expression of two or more gene products in cells is altered. The present invention also provides cell lines from such cells.

In one aspect, the invention comprises one or more cells comprising a non-naturally occurring Cas9 CRISPR-Cas system containing one or more dead guide RNAs disclosed or produced by the methods or algorithms described herein. Provides multicellular organisms, including. In one aspect, the invention is a cell, cell line, or cell line containing a non-naturally occurring Cas9 CRISPR-Cas system containing one or more dead guide RNAs disclosed or produced by the methods or algorithms described herein. Provides products from multicellular organisms.

A further aspect of the invention is a system engineered for either overexpression of Cas9 or preferably knock-in of Cas9, such as cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenics. One or as described herein, optionally in combination with a gRNA containing one or more guides at a technical level in the art, in combination with a mouse). The use of gRNAs containing multiple dead guides. As a result, a single system (eg, transgenic animals, cells) can serve as the basis for multiple genetic modification in system / network biology. Thanks to the dead guide, this is now possible both in vitro, ex vivo, and in vivo.

For example, after Cas9 is provided, one or more dead gRNAs may be provided to induce multiple gene regulation, and preferably multiple bidirectional gene regulation. One or more dead gRNAs can be provided in spatially and temporally appropriate forms if necessary or desired (eg, tissue-specific introduction of Cas9 expression). Both gRNAs containing dead guides or gRNAs containing guides are equally effective, thanks to the provision (eg expression) of transgenic / inducible Cas9 to the cells, tissues and animals of interest. Similarly, a further aspect of the invention is with systems engineered for knockout of Cas9 CRISPR-Cas (eg, cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice). One or more deads combined, optionally as described herein or in combination with a gRNA containing one or more guides at a technical level in the art, as described herein. The use of gRNAs containing guides.

As a result, combining the dead guides as described herein with the CRISPR applications described herein and the CRISPR applications known in the art provides a highly efficient and accurate means of multiple screening of the system. Brought (eg network biology). Such screening allows, for example, to identify specific combinations of gene activity (eg, on / off combinations) for identifying genes involved in diseases, specifically gene-related diseases. A preferred application of such screening is cancer. Similarly, screening for the treatment of such diseases is included in the present invention. Cells or animals may be exposed to abnormal conditions that result in disease or disease-like effects. Candidate compositions are provided and can be screened for effects in the desired multiple environment. For example, a patient's cancer cells may be screened for which combination of genes kills the cancer cells, and this information is then used to establish appropriate treatments.

In one aspect, the invention provides a kit comprising one or more of the constituents described herein. The kit may include a dead guide as described herein, with or without a guide as described herein.

The structural information provided herein allows the study of dead gRNA interactions with the target DNA and Cas9, engineering the dead gRNA structure to optimize the functionality of the entire Cas9 CRISPR-Cas system. It can be changed or changed. For example, insertion of an adapter protein capable of binding RNA can prolong the loop of dead gRNA without colliding with the Cas9 protein. These adapter proteins can also recruit effector proteins or fusions that contain one or more functional domains.

In some preferred embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is SID, or a concatemer of SID (eg, SID4X). In some embodiments, the functional domain is an epigenetic modification domain, which provides an epigenetic modification enzyme. In some embodiments, the functional domain is the activation domain, which may be the P65 activation domain.

One aspect of the invention is that the above elements are included in a single composition or in individual compositions. These compositions can advantageously be applied to the host to elicit functional effects at the genomic level.

In general, dead gRNAs are modified to provide specific binding sites (eg, aptamers) to which adapter proteins containing one or more functional domains bind (eg, via fusion proteins). The modified dead gRNA is a function on the adapter protein in a spatial arrangement that favors the binding of the adapter protein when the dead gRNA forms a CRISPR complex (ie, Cas9 that binds to the dead gRNA and the target) and enables the attribution function. The domain is modified to be positioned. For example, if the functional domain is a transcriptional activator (eg, VP64 or p65), the transcriptional activator is placed in a spatial arrangement in which it can affect the transcription of the target. Similarly, the transcription repressor will be advantageously positioned to influence the transcription of the target, and the nuclease (eg, Fok1) will be advantageously positioned to cleave or partially cleave the target. It will be.

Those skilled in the art allow modifications to dead gRNAs that allow the binding of adapters + functional domains, but cannot properly position the adapters + functional domains (eg, due to steric hindrance within the three-dimensional structure of the CRISPR complex). Will understand that it is an unintended modification. One or more modified dead gRNAs may be modified in tetraloop, stemloop 1, stemloop 2, or stemloop 3 as described herein, preferably either tetraloop or stemloop 2. Or, most preferably, it may be modified with both tetraloop and stemloop 2.

As described herein, functional domains include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity. , And one or more domains from the group consisting of molecular switches (eg, photoinducible). In some cases it is advantageous to provide at least one more NLS. In some cases, it is advantageous to position the NLS at the N-terminus. When two or more functional domains are included, they may be the same or different.

Dead gRNAs can be designed to contain multiple binding recognition sites (eg, aptamers) that are specific for the same or different adapter proteins. Dead gRNAs can be designed to bind to the promoter region upstream of -1000 to +1 nucleic acids, preferably -200 nucleic acids, at the transcription initiation site (ie, TSS). Positioning in this way improves the functional domains that affect gene activation (eg, transcriptional activators) or gene inhibition (eg, transcriptional repressors). A modified dead gRNA is one or more target loci contained in the composition (eg, at least one gRNA, at least two gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least It may be one or more modified dead gRNAs that target (30 gRNAs, at least 50 gRNAs).

An adapter protein is a protein that binds to an aptamer or recognition site introduced into a modified dead gRNA, and when the dead gRNA is incorporated into the CRISPR complex, one or more functional domains are appropriately positioned and assigned. It can be any protein whose function affects the target. As described in detail in the present application, this may be a coat protein, preferably a bacteriophage coat protein. Functional domains that associate with such adapter proteins (eg, in the form of fusion proteins) include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage. It may include one or more domains from the group consisting of activity, nucleic acid binding activity, and molecular switches (eg, photoinducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. If the functional domain is a transcription activator or transcription repressor, it is advantageous that at least NLS is preferably provided at the N-terminus. When two or more functional domains are included, they may be the same or different. The adapter protein can utilize known linkers to add such functional domains.

Thus, modified dead gRNAs, (inactivated) Cas9 (with or without functional domains), and binding proteins with one or more functional domains are each individually included in the composition and individually for the host. Alternatively, they can be administered in bulk. Alternatively, these components may be provided in a single composition for administration to a host. Administration to the host can be performed using viral vectors known to those of skill in the art for delivery to the host or described herein (eg, lentivirus vector, adenovirus vector, AAV vector). As described herein, the use of different selectable markers (eg, for lentiviral gRNA selection) and gRNA concentrations (eg, depending on whether multiple gRNAs are used) can improve efficacy. It can be advantageous to produce.

Based on this concept, several variations are appropriate for inducing genomic locus events, including DNA cleavage, gene activation, or gene deactivation. One of skill in the art can use the provided compositions to advantageously and specifically target single or multiple loci in the same or different functional domains to induce one or more genomic locus events. it can. The composition establishes cell lines and transgenic animals for screening in cell libraries and in vivo functional modeling (eg, identification of gene activation and function of lincRNA; functional acquisition modeling; loss of function modeling; optimization and screening purposes). Can be applied to a wide variety of methods for use of the compositions of the present invention).

The present invention includes the use of the compositions of the present invention for establishing and utilizing conditional or inducible CRISPR transgenic cells / animals that are not considered to precede the present invention or the present application. For example, a vector in which the target cell conditionally or inducibly contains Cas9 (eg, in the form of a Cre-dependent construct) and / or the adapter protein conditionally or inducibly and is introduced into the target cell. Upon expression, the vector is expressed to induce Cas9 expression and / or adapter expression in the target cell, or to give rise to that condition. Inducible genomic events that are affected by the functional domain by applying the teachings and compositions of the invention along with known methods of creating CRISPR complexes are also aspects of the invention. An example of this is the production of CRISPR knock-in / conditional transgenic animals (eg, mice containing, for example, a Lux-termination-poly A-Lox (LSL) cassette), and subsequently as described herein (eg,). One or more modified dead gRNAs (eg, from -200 nucleotides to TSS of the target gene of interest for gene activation), such as a coat protein, eg, a modified dead gRNA having one or more aptamers recognized by MS2. One or more adapter proteins as described herein (MS2-binding proteins linked to one or more VP64s) and means of inducing conditional animals (eg, Cre recombinases for inducing Cas9 expression). Is the delivery of one or more compositions that provide. Alternatively, the adapter protein may be provided as a conditional or inducible element with the conditional or inducible Cas9 to provide a model that is effective for screening purposes, which is advantageous for a wide variety of applications. In contrast, it requires minimal design and administration of specific dead gRNAs.

In another embodiment, the dead guide is further modified to improve specificity. A protected dead guide may be synthesized, which introduces a secondary structure at the 3'end of the dead guide and improves its specificity. A protected guide RNA (pgRNA) comprises a guide sequence and a protected strand that can hybridize to a target sequence at the target genomic locus of the cell, where the protected strand is optionally complementary to the guide sequence. And the guide sequence may be partially hybridizable with the protective strand. The pgRNA optionally contains an extension sequence. The thermodynamics of pgRNA-target DNA hybridization depends on the number of complementary bases between the guide RNA and the target DNA. "Thermodynamic protection" can be used to improve the specificity of dead gRNA by adding protected sequences. For example, one method is to add complementary protective strands of various lengths to the 3'end of the guide sequence in the dead gRNA. As a result, the protective strand binds to at least a portion of the dead gRNA, providing a protected gRNA (pgRNA). Similarly, the described embodiments can be used to readily protect the dead gRNA referred to herein to obtain a pgRNA. The protected strand may be either a separate RNA transcript or RNA strand or a chimeric version tethered to the 3'end of the dead gRNA guide sequence.

Use in tandem guides and multiple (tandem) targeting techniques We have shown that CRISPR enzymes as defined herein can use two or more RNA guides without loss of activity. Thereby, a single enzyme, system or complex as defined herein can be used to target multiple DNA targets, genes or loci to a CRISPR enzyme, system or complex as defined herein. It becomes possible to use. The guide RNA may be arranged in tandem or optionally separated by a nucleotide sequence such as direct repeat as defined herein. The positions of these different guide RNAs are tandem and do not affect activity. It is noted that the terms "CRISPR-Cas system", "CRISP-Cas complex", "CRISPR complex" and "CRISPR system" are used synonymously. The terms "CRISPR enzyme", "Cas enzyme", or "CRISPR-Cas enzyme" can also be used synonymously. In a preferred embodiment, the CRISPR enzyme, CRISP-Cas enzyme or Cas enzyme is Cas9, or any one of the modified or mutated variants described elsewhere herein. ..

In one aspect, the invention is a non-naturally occurring or engineered CRISPR enzyme used for tandem or multiple targeting, preferably a class 2 CRISPR enzyme, preferably a V-type or VI-type as described herein. CRISPR enzymes, eg, Cas9 as described elsewhere herein, without limitation. It should be understood that any of the CRISPR (or CRISPR-Cas or Cas) enzymes, complexes, or systems according to the invention as described elsewhere herein can be used in such techniques. is there. Any of the methods, products, compositions and uses as described elsewhere herein are equally applicable in the multiplex or tandem targeting techniques further detailed below. As further guidance, the following detailed embodiments and embodiments are provided.

In one aspect, the invention provides the use of Cas9 enzymes, complexes or systems as defined herein to target multiple loci. In one embodiment, it can be constructed by using multiple (tandem or multiple) guide RNA (gRNA) sequences.

In one aspect, the invention provides a method of using one or more elements of a Cas9 enzyme, complex or system as defined herein for tandem or multiple targeting, wherein the CRISPR system is plural. Contains a guide RNA sequence. Preferably, the gRNA sequence is separated by a nucleotide sequence, such as a direct repeat, as defined elsewhere herein.

Cas9 enzymes, systems or complexes as defined herein provide an effective means of modifying multiple target polynucleotides. Cas9 enzymes, systems or complexes as defined herein are modifications (eg, deletions, insertions, translocations, inactivations, activations) of one or more target polynucleotides in a large number of cell types. It has a wide variety of usefulness including. Thus, Cas9 enzymes, systems or complexes as defined herein, including targeting multiple loci within a single CRISPR system, include, for example, gene therapy, drug screening, disease diagnosis. , And has wide applicability in prognosis determination.

In one aspect, the invention targets a plurality of nucleic acid molecules, such as a Cas9 enzyme, system or complex as defined herein, i.e., a Cas9 protein associated with at least one destabilizing domain, and a DNA molecule. It provides a Cas9 CRISPR-Cas complex that comprises a plurality of guide RNAs to be converted, each of which has a plurality of guide RNAs that specifically target the corresponding nucleic acid molecule, eg, a DNA molecule. Each nucleic acid molecule target, eg, a DNA molecule, can encode a gene product or include a locus. By using a plurality of guide RNAs, it becomes possible to target a plurality of loci or a plurality of genes. In some embodiments, the Cas9 enzyme can cleave the DNA molecule that encodes the gene product. In some embodiments, the expression of the gene product is altered. Cas9 protein and guide RNA do not coexist in nature. The present invention includes a guide RNA containing a guide sequence arranged in tandem. The present invention further includes a coding sequence for the Cas9 protein whose codons have been optimized for expression in eukaryotic cells. In a preferred embodiment, the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell, and in a more preferred embodiment, the mammalian cell is a human cell. Expression of gene products can be reduced. The Cas9 enzyme may form part of a CRISPR system or complex, which can further specifically hybridize to a target sequence, each at the genomic locus of interest in the cell. A series of 2,3,4,5,6,7,8,9,10,15,25,25,30, or tandem-arranged guide RNAs (gRNAs) containing more than 30 guide sequences. Including. In some embodiments, the functional Cas9 CRISPR system or complex binds to multiple target sequences. In some embodiments, the functional CRISPR system or complex can edit multiple target sequences, eg, the target sequence may comprise a genomic locus, and in some embodiments gene expression. There can be changes. In some embodiments, the functional CRISPR system or complex may include additional functional domains. In some embodiments, the invention provides a method of altering or altering the expression of a plurality of gene products. The method may include the step of introducing into a cell containing or expressing the target nucleic acid, eg, a DNA molecule; eg, does the target nucleic acid encode a gene product? , Or can provide expression of a gene product (eg, regulatory sequence).

In a preferred embodiment, the CRISPR enzyme used for multiple targeting is Cas9, or the CRISPR system or complex comprises Cas9. In some embodiments, the CRISPR enzyme used for multiple targeting is AsCas9, or the CRISPR system or complex used for multiple targeting comprises AsCas9. In some embodiments, the CRISPR enzyme is LbCas9, or the CRISPR system or complex comprises LbCas9. In some embodiments, the Cas9 enzyme used for multiple targeting cleaves both strands of DNA to produce double-strand breaks (DSBs). In some embodiments, the CRISPR enzyme used for multiple targeting is nickase. In some embodiments, the Cas9 enzyme used for multiple targeting is dual nickase. In some embodiments, the Cas9 enzyme used for multiple targeting is a Cas9 enzyme, such as the DD Cas9 enzyme as defined elsewhere herein.

In embodiments, Cas9 may be paired, for example, as a pair of nickases, such as SaCas9 nickase (eSaCas9 nickase). In addition, Cas9 can be packaged in an AAV vector with one or more guides. This is Friedland AE et al, "Characteristics of Staphylococcus aureus Cas9; application of small Cas9 and paired nickase for all-in-one adeno-associated virus delivery (Charactration of Staphylococcus aureus Cas9: a smaller-genome Cas9). Associated virus delivery and pired nickase applications) ”, Genome Biol. 2015 Nov 24; 16: 257. It can be carried out as described in doi: 10.1186 / s13059-015-0817-8 (this disclosure is incorporated herein by reference).

In some common embodiments, the Cas9 enzyme used for multiple targeting is associated with one or more functional domains. In some more specific embodiments, the CRISPR enzyme used for multiple targeting is dead Cas9 as defined elsewhere herein.

In some embodiments, the invention provides a means of delivering a Cas9 enzyme, system or complex or polynucleotide as defined herein for use in multitargeting as defined herein. Non-limiting examples of such means of delivery include, for example, one or more particles delivering one or more components of a complex, one or more polynucleotides discussed herein (eg, one or more polynucleotides). One or more vectors comprising a CRISPR enzyme and providing a nucleotide encoding a CRISPR complex). In some embodiments, the vector may be a plasmid or viral vector, such as AAV, or a lentivirus. In particular, given the size limitation of AAV and the ability of Cas9 to fit in AAV while reaching the upper limit with additional guide RNA, transient transfection with a plasmid, eg, into HEK cells, may be advantageous.

Also provided are models that constitutively express the Cas9 enzyme, complex or system as used herein for multiple targeting. The organism may be transgenic, transfected with the vector, or progeny of such transfected organism. In a further aspect, the invention provides a composition comprising a CRISPR enzyme, system and complex as defined herein or a polynucleotide or vector described herein. Also provided is a Cas9 CRISPR system or complex containing multiple guide RNAs, preferably in a tandem arrangement format. The different guide RNAs may be separated by a nucleotide sequence such as direct repeat.

Also provided is a method of treating a subject, eg, a subject in need thereof, the method of which is the subject with a polynucleotide encoding the Cas9 CRISPR system or complex or any polynucleotide or vector described herein. Includes the step of inducing gene editing by transforming and administering them to a subject. Suitable repair templates may also be provided, which are delivered, for example, by a vector containing the repair template. Also provided is a method of treating a subject, eg, a subject in need thereof, the method of transcriptional activity of multiple target loci by transforming the subject with the polynucleotides or vectors described herein. The polynucleotide or vector comprises a step of inducing transformation or inhibition, preferably encoding or comprising a Cas9 enzyme, complex or system containing multiple guide RNAs located in tandem. It will be appreciated that the term "subject" can be replaced by the phrase "cell or cell culture" if any treatment is performed in exobibo, eg in cell culture.

Cas9 enzymes, complexes or systems containing multiple guide RNAs, preferably located in tandem, used in therapeutic methods as defined elsewhere herein are included or preferably located in tandem. Also provided is a composition comprising a polynucleotide or vector encoding or containing said Cas9 enzyme, complex or system comprising a plurality of guide RNAs. A kit of parts containing such a composition may be provided. The use of said compositions in the manufacture of pharmaceuticals for such therapeutic methods is also provided. The use of the Cas9 CRISPR system in screening, eg, function acquisition screening, is also provided by the present invention. Cells that are artificially forced to overexpress a gene can down-regulate (regain equilibrium) the gene over time, for example by a negative feedback loop. By the start of screening, unregulated genes can be reduced again. Inducible Cas9 activators allow transcription to be induced just prior to screening, thus minimizing the possibility of false negative hits. Therefore, the possibility of false negative results can be minimized by using the present invention in screening, such as function acquisition screening.

In one aspect, the invention provides an engineered, non-naturally occurring CRISPR system that includes a Cas9 protein and multiple guide RNAs, each of which specifically targets a DNA molecule encoding an intracellular gene product. , Thus, multiple guide RNAs each target their specific DNA molecule encoding the gene product, and the Cas9 protein cleaves the target DNA molecule encoding the gene product, thereby altering the expression of the gene product. And here the CRISPR protein and guide RNA do not co-exist in nature. The present invention includes a plurality of guide RNAs comprising a plurality of guide sequences, preferably separated by a nucleotide sequence such as direct repeat and optionally fused with a tracr sequence. In certain embodiments of the invention, the CRISPR protein is a V-type or VI-type CRISPR-Cas protein, and in a more preferred embodiment the CRISPR protein is a Cas9 protein. The present invention further includes Cas9 proteins whose codons have been optimized for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

In another embodiment, the invention encodes a CRISPR protein with a first regulatory element operably linked to multiple Cas9 CRISPR-based guide RNAs, each of which specifically targets a DNA molecule encoding a gene product. Provided is an engineered non-naturally occurring vector system comprising one or more vectors comprising a second operably linked regulatory element. Both regulatory elements may be located on the same vector of the system or on different vectors. Multiple guide RNAs can target multiple DNA molecules encoding multiple gene products in the cell, and the CRISPR protein can cleave multiple DNA molecules encoding the gene product (which can be one or both). The strands can be cleaved or have virtually no nuclease activity), thereby altering the expression of multiple gene products; and where the CRISPR protein and multiple guide DNAs coexist in nature. do not do. In a preferred embodiment, the CRISPR protein is an optionally codon-optimized Cas9 protein for expression in eukaryotic cells. In a preferred embodiment, the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, the expression of each of the plurality of gene products is altered, preferably reduced.

In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system is (a) a direct repeat sequence and one or more for inserting one or more guide sequences upstream or downstream (whichever is applicable) of the direct repeat sequence. A first regulatory element operably linked to the insertion site (one or more guide sequences, when expressed, leads to sequence-specific binding of the CRISPR complex to one or more target sequences in eukaryotic cells. , The CRISPR complex comprises a Cas9 enzyme complexed with one or more guide sequences that hybridize to one or more target sequences); and (b) preferably at least one nuclear localization sequence and / Or include a second regulatory element operably linked to an enzyme coding sequence encoding said Cas9 enzyme, comprising at least one NES; where components (a) and (b) are the same or different in the system. Located on the vector. If applicable, tracr sequences may also be provided. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, and each of these two or more guide sequences is expressed. , Leads to sequence-specific binding of the Cas9 CRISPR complex to different target sequences in eukaryotic cells. In some embodiments, the CRISPR complex has one or more nuclear localizations of sufficient intensity to drive the accumulation of a detectable amount of the Cas9 CRISPR complex in or out of the nucleus of eukaryotic cells. Includes a chemical sequence and / or one or more NES. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, each of the guide sequences is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides in length.

The recombinant expression vector is selected based on the host cell used for expression in a form suitable for expression of the nucleic acid in the host cell, that is, the recombinant expression vector is operably linked to the nucleic acid sequence to be expressed. Containing a polynucleotide encoding a Cas9 enzyme, system or complex used for multitargeting as defined herein (meaning that it comprises one or more regulatory elements obtained). Within a recombinant expression vector, "operably linked" allows expression of nucleotide sequences (eg, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell). It is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements.

In some embodiments, the host cell is transiently or populated with one or more vectors containing a polynucleotide encoding a Cas9 enzyme, system or complex used for multitargeting as defined herein. Transfected non-transiently. In some embodiments, the cell is transfected as it is naturally present in the subject. In some embodiments, the cells to be transfected are harvested from the subject. In some embodiments, the cells are derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art and are exemplified elsewhere herein. Cell lines are available from a variety of sources known to those of skill in the art (see, eg, the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, using cells transfected with one or more vectors containing a polynucleotide encoding a Cas9 enzyme, system or complex used for multitargeting as defined herein. A novel cell line containing one or more vector-derived sequences is established. In some embodiments, the components of the Cas9 CRISPR system or complex used for multitargeting as described herein (by transient transfection of one or more vectors, or transfection with RNA). Using cells that have been transiently transfected and modified by the activity of the Cas9 CRISPR system or complex (such as), novel cell lines that include cells that contain modifications but lack any other exogenous sequence. Is established. In some embodiments, transiently or non-transiently, one or more vectors containing a polynucleotide encoding a Cas9 enzyme, system or complex used for multitargeting as defined herein. One or more test compounds are evaluated using the transfected cells, or cell lines derived from such cells.

The term "regulatory element" is as defined elsewhere herein.

Advantageous vectors include lentivirus and adeno-associated virus, and the type of such vector can also be selected to target a particular type of cell.

In one aspect, the invention comprises (a) a direct repeat sequence and one or more insertion sites for inserting one or more guide RNA sequences upstream or downstream (whichever is applicable) of the direct repeat sequence. A first regulatory element operably linked to and (one or more guide sequences, when expressed, sequence-specific for the Cas9 CRISPR complex to each one or more target sequences in eukaryotic cells. The Cas9 CRISPR complex, wherein the Cas9 CRISPR complex comprises a Cas9 enzyme complexed with one or more guide sequences that hybridize to each one or more target sequences); and / or (b) preferably. Provides a eukaryotic host cell comprising a second regulatory element operably linked to an enzyme coding sequence encoding the Cas9 enzyme, including at least one nuclear localization sequence and / or NES. In some embodiments, the host cell comprises components (a) and (b). If applicable, tracr sequences may also be provided. In some embodiments, the components (a), components (b), or components (a) and (b) are stably integrated into the genome of the host eukaryotic cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first adjusting element and optionally separated by direct repeat, wherein the component (a) further comprises. Each of these two or more guide sequences, when expressed, leads to sequence-specific binding of the Cas9 CRISPR complex to different target sequences in eukaryotic cells. In some embodiments, the Cas9 enzyme is one or more nuclear stations of sufficient intensity to drive the accumulation of a detectable amount of the CRISPR enzyme in and / or out of the nucleus of eukaryotic cells. Includes a resident sequence and / or a nuclear localization sequence or NES.

In some embodiments, the Cas9 enzyme is a V-type or VI-type CRISPR-based enzyme. In some embodiments, the Cas9 enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Francisella tularensis 1, Francisella tularensis subsp. Novicida, Prebotera albensis (Prevotella) bacteria Lactera lancera Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp.) BV3L6 , Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens (Eubacterium eligens), Moraxera eligens, Moraxera libera Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevoterella disiens (Prevoterla diciens), or Porphyromacac. Can include further changes or mutations in Cas9 as defined in, and may be chimeric Cas9. In some embodiments, the Cas9 enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the one or more guide sequences (each) are at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides in length. .. When multiple guide RNAs are used, they are preferably separated by direct repeat sequences. In some embodiments, the invention provides a non-human eukaryote, preferably a multicellular eukaryote, comprising a eukaryotic host cell according to any of the described embodiments. In another aspect, the invention provides a eukaryote comprising a eukaryotic host cell according to any of the described embodiments; preferably a multicellular eukaryote. The organism in some embodiments of these embodiments may be an animal; eg, a mammal. In addition, the organism may be an arthropod such as an insect. The organism may also be a plant. In addition, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more of the constituents described herein. In some embodiments, the kit includes a vector system and instructions for use of the kit. In some embodiments, the vector system is (a) a direct repeat sequence and one or more for inserting one or more guide sequences upstream or downstream (whichever is applicable) of the direct repeat sequence. A first regulatory element operably linked to the insertion site (the guide sequence, when expressed, leads to sequence-specific binding of the Cas9 CRISPR complex to the target sequence in eukaryotic cells, where the Cas9 CRISPR complex Includes a Cas9 enzyme complexed with a guide sequence that hybridizes to the target sequence); and / or (b) operably linked to an enzyme coding sequence encoding said Cas9 enzyme containing a nuclear localization sequence. Also includes a second adjustment element. If applicable, tracr sequences may also be provided. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it leads to sequence-specific binding of the CRISPR complex to different target sequences in eukaryotic cells. In some embodiments, the Cas9 enzyme comprises one or more nuclear localization sequences that are strong enough to drive the accumulation of a detectable amount of the CRISPR enzyme in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a V-type or VI-type CRISPR-based enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Francisella tularensis 1, Francisella tularensis subsp. Novicida, Prebotera albensis (Prevotella) bacteria Lactera lancera Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp.) BV3L6 , Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens (Eubacterium eligens), Moraxera eligens, Moraxera libera Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevoterella disiens (Prevoterella disiens) or at least one Domae diaPorca dia Derived from (modified to associate with it), it can contain further alterations or mutations in Cas9, and may be chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially lacks DNA strand cleavage activity (eg, compared to wild-type enzymes or enzymes that do not have mutations or alterations that reduce nuclease activity. Nuclease activity of 5% or less). In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides in length.

In one aspect, the invention provides a method of modifying multiple target polynucleotides in a host cell, such as a eukaryotic cell. In some embodiments, the method involves binding the Cas9CRISPR complex to a plurality of target polynucleotides, eg, causing cleavage of the plurality of target polynucleotides, thereby modifying the plurality of target polynucleotides. Included, where the Cas9CRISPR complex comprises a Cas9 enzyme complexed with a plurality of guide sequences, each hybridizing to a particular target sequence within the target polynucleotide, the plurality of guide sequences linked to a direct repeat sequence. Has been done. Where applicable, tracr sequences can also be provided (eg, thereby providing a single guide RNA, sgRNA). In some embodiments, the cleavage comprises cleaving one or two strands at each position of the target sequence with the Cas9 enzyme. In some embodiments, the cleavage results in reduced transcription of multiple target genes. In some embodiments, the method further comprises the step of repairing one or more of the cleaved target polynucleotides by homologous recombination with an exogenous template polynucleotide, wherein the repair is one of the target polynucleotides. It results in mutations involving the insertion, deletion, or substitution of one or more nucleotides in one or more. In some embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene that contains one or more of the target sequences. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, where the one or more vectors are linked to a Cas9 enzyme and a plurality of direct repeat sequences. Drives the expression of one or more of the guide RNA sequences. If applicable, tracr sequences may also be provided. In some embodiments, the vector is delivered to a subject eukaryotic cell. In some embodiments, the modification occurs in the eukaryotic cell in a cell culture. In some embodiments, the method further comprises the step of isolating the eukaryotic cell from the subject prior to the modification. In some embodiments, the method further comprises the step of returning the eukaryotic cell and / or cells derived thereto to the subject.

In one aspect, the invention provides a method of modifying the expression of multiple polynucleotides in eukaryotic cells. In some embodiments, the method comprises binding the Cas9 CRISPR complex to a plurality of polynucleotides, which results in increased or decreased expression of the polynucleotide; where the Cas9 CRISPR complex is: , The Cas9 enzyme complexed with a plurality of guide sequences, each of which specifically hybridizes to a unique target sequence within the polynucleotide, is linked to a direct repeat sequence. If applicable, tracr sequences may also be provided. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, where the one or more vectors are linked to a Cas9 enzyme and a plurality of direct repeat sequences. Drives expression of one or more of the guide sequences. If applicable, tracr sequences may also be provided.

In one embodiment, the invention provides a recombinant polynucleotide comprising a plurality of guide RNA sequences upstream or downstream (whichever is applicable) of a direct repeat sequence, wherein each of the guide sequences is expressed. It leads to sequence-specific binding of the Cas9CRISPR complex to its corresponding target sequence present in eukaryotic cells. In some embodiments, the target sequence is a viral sequence that is present in eukaryotic cells. If applicable, tracr sequences may also be provided. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

Aspects of the invention are described herein which may include a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a target genomic locus of a cell and at least one or more nuclear localized sequences. Includes non-naturally occurring or engineered compositions that may include Cas9 enzymes as defined.

One aspect of the invention includes a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing any of the compositions described herein into the cell.

One aspect of the invention is that the above elements are included in a single composition or in individual compositions. These compositions can advantageously elicit functional effects at the genomic level when applied to the host.

As used herein, the term "guide RNA" or "gRNA" has the meaning as used elsewhere in the specification (leaning) and hybridizes with the target nucleic acid sequence to target nucleic acid. Includes any polynucleotide sequence that has sufficient complementarity with the target nucleic acid sequence to derive sequence-specific binding of the nucleic acid targeting complex to the sequence. Each gRNA can be designed to contain multiple binding recognition sites (eg, aptamers) that are specific for the same or different adapter proteins. Each gRNA can be designed to bind to the promoter region upstream of -1000 to +1 nucleic acid, preferably -200 nucleic acid, at the transcription initiation site (ie, TSS). Positioning in this way improves the functional domains that affect gene activation (eg, transcriptional activators) or gene inhibition (eg, transcriptional repressors). The modified gRNA is one or more modified gRNAs that target one or more target loci contained in the composition (eg, at least one gRNA, at least two gRNAs, at least five gRNAs). , At least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs). The plurality of gRNA sequences are arranged in tandem and are preferably separated by direct repeat.

Therefore, the gRNA and CRISPR enzyme as defined herein are each individually contained in the composition and can be administered to the host individually or collectively. Alternatively, these components may be provided in a single composition for administration to a host. Administration to the host can be carried out via viral vectors known to those of skill in the art for delivery to the host or described herein (eg, lentivirus vector, adenovirus vector, AAV vector). As described herein, the use of different selectable markers (eg, for lentivirus sgRNA selection) and gRNA concentrations (eg, depending on whether multiple gRNAs are used) can improve efficacy. It can be advantageous to produce. Based on this concept, several variations are appropriate for inducing genomic locus events, including DNA cleavage, gene activation, or gene deactivation. One of ordinary skill in the art will use the provided composition to advantageously and specifically target single or multiple loci having the same or different functional domains to induce one or more genomic locus events. be able to. The composition comprises screening in a library of cells and in vivo functional modeling (eg, gene activation and functional identification of lincRNA; functional acquisition modeling; loss of function modeling; cell lines and transgenic animals for optimization and screening purposes. It can be applied to a wide variety of methods for the use of the compositions of the present invention to establish.

The present invention includes the use of the compositions of the present invention for establishing and utilizing conditioned or inducible CRISPR transgenic cells / animals; eg, Platt et al. , Cell (2014), 159 (2): 440-455, or PCT patent gazettes cited herein, such as WO 2014/093622 (PCT / US2013 / 074667). .. For example, cells or animals such as non-human animals such as vertebrates or mammals such as rodents such as mice, rats, or other laboratory or field animals such as cats, dogs, sheep, etc. are "knocked in". Also, the animal thereby expresses Cas9 conditionally or inducibly, as in Platt et al. Thus, the target cell or animal contains the CRISPR enzyme (eg Cas9) conditionally or inducibly (eg in the form of a Cre-dependent construct), and upon expression of the vector introduced into the target cell, the vector will be CRISPR in the target cell. It is expressed to induce or give rise to the expression of an enzyme (eg, Cas9). Inducible genomic events are also aspects of the invention by applying the teachings and compositions as defined herein along with known methods of creating CRISPR complexes. Examples of such inducible events are described elsewhere herein.

In some embodiments, especially when a hereditary disease is targeted in a therapeutic method, the phenotypic change is preferably the result of genomic modification, preferably to modify or alter the phenotype here. A repair template is provided.

In some embodiments, potentially targeted diseases include those associated with disease-causing splice deficiency.

In some embodiments, cell targets include hematopoietic stem / progenitor cells (CD34 +); human T cells; and eyes (retinal cells) -eg, photoreceptive progenitor cells.

In some embodiments, the gene target is human β-globin-HBB, including for the treatment of sickle cell anemia, by stimulation of gene conversion (using a closely related HBD gene as an endogenous template); CD3 (T cells); and CEP920-retina (eye).

In some embodiments, disease targets also include cancers that cause splice deficiency; sickle cell anemia (based on point mutations); HBV, HIV; β-thalassemia; and ophthalmic or eye diseases-eg, label congenital. Amaurosis (LCA)-also includes. In some embodiments, delivery methods include cationic lipid-mediated "direct" delivery of enzyme-guide complexes (ribonuclear proteins) and electroporation of plasmid DNA.

The methods, products and uses described herein can be used for non-therapeutic purposes. In addition, any of the methods described herein can be applied in vitro and in exobibo.

In some embodiments
I. Two or more CRISPR-Cas-based polynucleotide sequences
(A) A first guide sequence capable of hybridizing to a first target sequence at a polynucleotide locus,
(B) A second guide sequence capable of hybridizing to a second target sequence at the polynucleotide locus,
(C) Direct repeat array,
Polynucleotide sequence containing, and II. Non-naturally occurring or engineered compositions comprising the Cas9 enzyme or a second polynucleotide sequence encoding it are provided.
When transcribed, the first and second guide sequences lead to sequence-specific binding of the first and second Cas9 CRISPR complexes to the first and second target sequences, respectively.
The first CRISPR complex comprises a Cas9 enzyme complexed with a first guide sequence capable of hybridizing to a first target sequence.
The second CRISPR complex contains a Cas9 enzyme that has formed a complex with a second guide sequence that is hybridizable to the second target sequence.
The first guide sequence leads to the cleavage of one strand of the DNA duplex near the first target sequence, and the second guide sequence leads to the cleavage of the other strand near the second target sequence. Dechaining is induced, thereby altering an organism or non-human or non-animal organism. Similarly, assume, for example, a composition each specific to one target and as described herein or a composition containing more than two guide RNAs tandemly located in a CRISPR system or complex. can do.

In another embodiment Cas9 is delivered to cells as a protein. In another particularly preferred embodiment, Cas9 is delivered to cells as a protein or as a nucleotide sequence encoding it. Delivery to cells as a protein may include delivery of a ribonucleoprotein (RNP) complex, where the protein forms a complex with multiple guides.

In certain embodiments, host cells and cell lines that are modified by or contain the compositions, systems or modifying enzymes of the invention, including stem cells and their progeny, are provided.

In some embodiments, a method of cell therapy is provided, wherein, for example, a single cell or cell population is sampled or cultured, wherein the one or more cells are exobibo as described herein. It has been modified or modified and then reintroduced (sampled cells) or introduced (cultured cells) into the organism. Stem cells, whether embryonic stem cells or induced pluripotent or totipotent stem cells, are particularly preferred in this regard. But, of course, in vivo embodiments are also envisioned.

The methods of the invention can further include delivery of a template such as a repair template, which may be dsODN or ssODN (see below). Delivery of the template may be simultaneous or separate with delivery of some or all of the CRISPR enzyme or guide RNA, and may be by the same or different delivery mechanisms. In some embodiments, the template is preferably delivered with the guide RNA, preferably with the CRISPR enzyme. One example may be an AAV vector, where the CRISPR enzyme is AsCas9 or LbCas9.

The method of the present invention is (a) a step of delivering a double-stranded oligodeoxynucleotide (dsODN) containing an overhang complementary to the overhang caused by the double-strand break to cells, wherein the dsODN is an object. Incorporating into the locus of the gene; or-(b) delivering a single-stranded oligodeoxynucleotide (ssODN) to cells, wherein the ssODN acts as a template for homology-dependent repair of the double-stranded break. Can be further included. The method of the present invention may be a method of preventing or treating a disease of an individual, and the disease is optionally caused by a defect in the locus of interest. The method of the present invention can be performed in vivo in an individual or ex vivo on cells taken from an individual, and the cells are optionally returned to the individual.

The present invention is also obtained using the CRISPR enzyme or Cas enzyme or Cas9 enzyme or CRISPR-CRISPR enzyme or CRISPR-Cas or CRISPR-Cas9 system used for tandem or multitargeting as defined herein. Also includes products.

In one aspect of an escorted guide for the Cas9 CRISPR-Cas system according to the present invention, the present invention provides such a system involving an escorted Cas9 CRISPR-Cas system or complex, particularly an escorted Cas9 CRISPR-Cas system guide. “With escort” means that the Cas9 CRISPR-Cas system or complex or guide is delivered intracellularly at a selected time or place, so that the activity of the Cas9 CRISPR-Cas system or complex or guide is spatial or temporal. Means to be controlled by. For example, the activity and destination of the Cas9 CRISPR-Cas system or complex or guide can be regulated by an escort RNA aptamer sequence that has a binding affinity for an aptamer ligand such as a cell surface protein or other localized cellular component. Alternatively, the escort aptamer may respond to an on-cell or intracellular aptamer effector, such as a transient effector, such as an external energy source applied to the cell at a particular point in time.

An escorted Cas9 CRISPR-Cas system or complex has a gRNA having a functional structure designed to improve the structure, architecture, stability, gene expression, or any combination thereof of the gRNA. Such a structure may include an aptamer.

Aptamers, for example, use a technique called in vitro evolution (SELEX; Tuerk C, Gold L: "In vitro evolution: RNA ligand for Bacterophage T4 DNA polymerase (Sythematic evolution of ligands by exponential evolution: RNA)". T4 DNA polymerase) ”. Science 1990, 249: 505-510), a biomolecule that can be designed or selected to bind tightly to other ligands. Nucleic acid aptamers can be selected, for example, from a pool of random sequence oligonucleotides, have high binding affinity and specificity for a wide range of biomedically relevant targets, and have a wide range of therapeutic utility of aptamers. (Keefe, Anthony D., Supriya Pai, and And Andrew Ellington. "Aptamers as therapeutics". Nature Review (37) Dev5: 7) Drug10 (Drug10). These features also suggest a wide range of uses for aptamers as drug delivery media (Levi-Nissenbaum, Etgar, et al. "Nanotechnology and aptamers: applications in drug". ). . J Clin Invest 2000, 106: 923-928). RNA aptamers that bind to the fluorophore and mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R. Jaffrey. "RNA mimics of green fluorescent protein ) ”. Science 333.6042 (2011): 642-646), an aptamer acting as a molecular switch that responds to a que by changing its properties can also be constructed. It has also been suggested that aptamers can be used, for example, as components of targeted siRNA therapeutic delivery systems that target cell surface proteins (Zhou, Jiehua, and John J. Rossi. "Aptamer-targeted cell-specific RNA". Interference (Aptamer-targeted cell-specific RNA interference) ”.Silence 1.1 (2010): 4).

Thus, the specification has been modified by one or more aptamers designed to improve gRNA delivery, including, for example, cross-cell membrane delivery, intracellular compartment delivery, or intracellular delivery. gRNA is provided. Such a structure comprises a portion for making the guide deliverable, inducible or responsive to a selected effector in addition to or without such one or more aptamers. obtain. Accordingly, the present invention includes, without limitation, pH, hypoxia, O ₂ concentration, temperature, protein concentration, enzyme concentration, lipid structures, exposure, mechanical disruption (e.g., ultrasound), magnetic, electric fields, or electromagnetic radiation Includes gRNAs that respond to normal or pathological physiological conditions.

One aspect of the present invention is
An RNA-guided sequence that can hybridize to a target sequence at the target genomic locus of the cell;
Provided is a non-naturally occurring or engineered composition comprising an escorted guide RNA (egRNA) comprising an escort RNA aptamer sequence, the escort aptamer having binding affinity for intracellular or intracellular aptamer ligands. However, or this escort aptamer is responsive to intracellular or intracellular localized aptamer effectors, where the presence of intracellular or intracellular aptamer ligands or effectors is spatially or temporally restricted.

Escort aptamers can, for example, change conformations in response to interactions with intracellular aptamer ligands or effectors.

Escort aptamers can have a specific binding affinity for aptamer ligands.

The aptamer ligand can be localized at a location or compartment of the cell, eg, on or within the membrane of the cell. Therefore, when an escort aptamer binds to an aptamer ligand, gRNA is guided to a target position in the cell, such as inside the cell, via binding to the aptamer ligand, which is a cell surface ligand. In this way, various spatially localized locations within the cell, such as the cell nucleus or mitochondria, can be targeted.

After the intended changes have been introduced, such as by editing the intended copy of the gene in the cell's genome, CRISPR / Cas9 expression does not need to continue further in the cell. In fact, sustained expression may not be desirable in some cases, such as when off-target effects occur at unintended genomic sites. Therefore, timed expression can be useful. Inducible expression provides one approach, but in addition, Applicants have engineered a self-inactivating Cas9 CRISPR-Cas system that relies on the use of non-coding guide target sequences within the CRISPR vector itself. Thus, after initiation of expression, the CRISPR system can result in its own disruption, but before the disruption is complete, it may have time to edit the genomic copy of the target gene (normal point mutations in diploid cells). So, this requires at most two edits). Simply, the self-activating Cas9 CRISPR-Cas system targets the coding sequence of the CRISPR enzyme itself, or one or more non-coding guide targets complementary to the unique sequence present in one or more of the following: Includes additional RNA (ie, guide RNA) that targets the sequence: (a) within the promoter driving the expression of the non-coding RNA element, (b) within the promoter driving the expression of the Cas9 gene, (c) the Cas9 coding sequence. Within 100 bp from the ATG translation initiation codon in (d) within the reverse terminal repeat sequence (iTR) of the virus delivery vector, eg, in the AAV genome.

The egRNA may include an RNA aptamer ligation sequence that operably links the escort RNA sequence to the RNA guide sequence.

In embodiments, the egRNA may contain one or more photodissociative bonds or non-naturally occurring residues.

In one embodiment, the escort RNA aptamer sequence may be complementary to the target miRNA, which may or may not be present in the cell, and thus the escort RNA aptamer to the target miRNA only when the target miRNA is present. There is sequence binding, which results in cleavage of the eggRNA by RNA-induced silencing complex (RISC) in the cell.

In embodiments, the escort RNA aptamer sequence may be, for example, 10 to 200 nucleotides in length, and the egRNA may comprise two or more escort RNA aptamer sequences.

It should be understood that any of the RNA-guided sequences as described elsewhere herein can be used for the egRNAs described herein. In certain embodiments of the invention, the guide RNA or mature crRNA comprises, consists of, or consists of a direct repeat sequence and a guide sequence or spacer sequence. In certain embodiments, the guide RNA or mature crRNA comprises, is, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence. In certain embodiments, the guide RNA or mature crRNA comprises 19 nt of partial direct repeats, followed by 23-25 nt of guide or spacer sequences. In certain embodiments, the effector protein is the FnCas9 effector protein, which requires at least 16 nt of guide sequences to achieve detectable DNA cleavage and at least 17 nt to achieve efficient DNA cleavage in vitro. A guide array is required. In certain embodiments, the direct repeat sequence is located upstream (ie, 5'side) of the guide or spacer sequence. In a preferred embodiment, the seed sequence of the FnCas9 guide RNA (ie, an important sequence essential for sequence recognition and / or hybridization at the target locus) is the first approximately 5 nt on the 5'end of the guide sequence or spacer sequence. Is within.

The egRNA is, for example, at least 97% when compared to Cas9 without at least one mutation, such as Cas9 having less than 5% nuclease activity of Cas9 without at least one mutation, eg, at least one mutation. , Or may be included in a non-naturally occurring or engineered Cas9 CRISPR-Cas complex composition, with Cas9 which may contain a mutation with a 100% reduction in nuclease activity. Cas9 may also contain one or more nuclear localization sequences. Mutant Cas9 enzymes with regulated activity, such as reduced nuclease activity, are described elsewhere herein.

The engineered Cas9 CRISPR-Cas composition can be provided to cells such as eukaryotic cells, mammalian cells, or human cells.

In embodiments, the compositions described herein have at least three functional domains, at least one of which is associated with Cas9 and at least two of which are associated with egRNA. Includes the Cas9 CRISPR-Cas complex.

The compositions described herein are genomic locus events in vivo to host cells such as eukaryotic cells, specifically mammalian cells, or non-human eukaryotes, specifically non-human mammals such as mice. Can be used for the introduction of. Genomic locus events can include affecting gene activation, gene inhibition, or cleavage at the locus. The compositions described herein can also be used to modify genomic loci of interest to alter gene expression in cells. Methods for introducing genomic locus events into host cells using the Cas9 enzyme provided herein are described in detail elsewhere herein. Delivery of the composition is also, for example, by delivery of one or more nucleic acid molecules encoding the composition, wherein the one or more nucleic acid molecules are operably linked to one or more regulatory sequences. Well, and in vivo expression of one or more nucleic acid molecules may be, for example, by lentivirus, adenovirus, or AAV.

The present invention provides compositions and methods capable of adapting gRNA-mediated gene editing activity. The present invention provides gRNA secondary structure that improves cleavage efficiency by increasing gRNA and / or increasing the amount of RNA delivered to cells. This gRNA may contain photodissociative or inducible nucleotides.

To increase the effectiveness of gRNAs, such as gRNAs delivered by viral or non-viral techniques, Applicants have added secondary structures within gRNAs that enhance their stability and improve gene editing. Separately, to eliminate the lack of effective delivery, Applicants modified gRNAs with cell-permeable RNA aptamers; these aptamers bind to cell surface receptors and promote the entry of gRNAs into cells. To do. In particular, cell-permeable aptamers can be designed to target specific cell receptors to mediate cell-specific delivery. Applicants have also created an inductive guide.

The photoreactivity of the inducible system can be achieved by activation and binding of cryptochrome-2 and CIB1. Blue light stimulation causes an activation conformational change in cryptochrome-2, which recruits its binding partner CIB1. This binding is fast and reversible, reaching saturation less than 15 seconds after pulse stimulation and returning to baseline less than 15 minutes after the end of stimulation. These rapid binding kinetics result in systems that are temporally constrained only by the rate of transcription / translation and transcription / proteolysis, rather than the uptake and clearance of the inducer. Cryptochrome-2 activation is also extremely sensitive, allowing the use of low light intensity stimuli and reducing the risk of phototoxicity. Moreover, in contexts such as the intact mammalian brain, variable light intensity can be used to control the size of the stimulus region, providing greater accuracy than vector delivery alone can provide.

The present invention contemplates that an energy source such as electromagnetic radiation, sound energy or thermal energy guides the guide. Advantageously, electromagnetic radiation is a component of visible light. In a preferred embodiment, the light is blue light with a wavelength of about 450-about 495 nm. In a particularly preferred embodiment, the wavelength is about 488 nm. In another preferred embodiment, the light stimulus is by a pulse. The optical power may be in the range of about 0-9 mW / cm ² . In a preferred embodiment, a low stimulation paradigm of 0.25 seconds every 15 seconds should result in maximum activation.

The cells involved in the practice of the present invention may be prokaryotic or eukaryotic cells, preferably animal cells, plant cells or yeast cells, more preferably mammalian cells.

A chemical or energy sensitive guide can cause a conformational change upon binding of a chemical source or induction by energy, which can act as a guide and allow the Cas9 CRISPR-Cas system or complex to function. Can be. The present invention applies a chemical source or energy to function the guide and the Cas9 CRISPR-Cas system or complex; and optionally further determine that the expression of the genomic locus is altered. May include.

This chemical inducible system has several different designs: 1. 2. ABI-PYL-based system inducible by abscisic acid (ABA) (see, eg, http: //stke.sciencemag.org/cgi/content/abstract/sigtrans; 4/164 / rs2). An FKBP-FRB-based system (eg, http: //www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html) that can be induced by rapamycin (or related chemicals based on rapamycin). See) 3. A GID1-GAI-based system that can be induced by gibberellin (GA) (see, eg, http://www.nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

Another system contemplated by the present invention is a chemical-induced system based on changes in intracellular localization. Applicants also have a genomic locus of interest in which the polypeptide comprises a DNA binding domain containing at least 5 or more transcriptional activator-like effector (TALE) monomers and is linked to at least 1 or more effector domains. We have also developed a system in which at least one or more semi-monomers specifically aligned to target a chemical or energy-sensitive protein are further linked. This protein results in a change in the intracellular localization of the entire polypeptide (ie, transport of the entire polypeptide from the cytoplasm of the cell into the nucleus) upon binding of the chemical or energy transfer to the chemical or energy sensitive protein. Become. Polypoly by this transport of the entire polypeptide from one intracellular compartment or organelle whose activity is blocked due to the absence of a substrate for the effector domain into another intracellular compartment or organelle in which the substrate is present. The entire peptide can be contacted with its desired substrate (ie, genomic DNA in the mammalian nucleus), resulting in activation or suppression of target gene expression.

This type of system can also be used to induce cleavage of the genomic locus of interest in the cell when the effector domain is a nuclease.

The chemical-inducible system may be an estrogen receptor (ER) -based system inducible by 4-hydroxytamoxifen (4OHT) (eg, http: //www.pnas.org/content/104/3). / 1027. See abstract). A mutant ligand-binding domain of the estrogen receptor called ERT2 translocates into the cell nucleus upon binding of 4-hydroxytamoxifen. In a further embodiment of the invention, any of any nuclear receptor, thyroid hormone receptor, retinoic acid receptor, estrogen receptor, estrogen-related receptor, glucocorticoid receptor, progesterone receptor, androgen receptor. Naturally occurring or engineered derivatives can be used in inducible systems similar to ER-based inducible systems.

Another inducible system is based on a design that uses a transient receptor potential (TRP) ion channel based system that can be induced by energy, heat or radio waves (eg, http://www.sciencemag.org/content/). See 336/6081/604). These TRP family proteins respond to a variety of stimuli, including light and heat. When this protein is activated by light or heat, ion channels open, allowing ions such as calcium to enter the cell membrane. This influx of ions binds to an intracellular ion interaction partner linked to the polypeptide containing the guide and other components of the Cas9 CRISPR-Cas complex or system, and this binding alters the intracellular localization of the polypeptide. It will induce the invasion of the entire polypeptide into the cell nucleus. Once inside the nucleus, the guide protein and other components of the Cas9 CRISPR-Cas complex become active and regulate target gene expression in cells.

This type of system can also be used to induce cleavage of the genomic locus of interest in the cell; and in this regard, it is noted that the Cas9 enzyme is a nuclease. Light can be generated by a laser or other form of energy source. Heat can be generated by a temperature rise generated by an energy source or by nanoparticles that release heat after absorption of energy from an energy source delivered in the form of radio waves.

Photoactivation can be an advantageous embodiment, but sometimes this can be disadvantageous, especially for in vivo applications where light may not penetrate the skin or other organs. In this case, other energy activation methods, in particular electric field energy and / or ultrasonic waves having similar effects are contemplated.

The electric field energy is preferably administered using one or more electrical pulses from about 1 volt / cm to about 10 kvolt / cm under in vivo conditions, substantially as described in the art. Instead of or in addition to the pulse, the electric field may be delivered continuously. The electrical pulse can be applied for 1 μs to 500 ms, preferably 1 μs to 100 ms. The electric field can be applied continuously or pulsedly for about 5 minutes.

As used herein, "electric field energy" is the electrical energy to which a cell is exposed. Preferably, the electric field has an intensity of about 1 volt / cm to about 10 kV / cm or more under in vivo conditions (see WO 97/49450).

As used herein, the term "electric field" includes exponential and / or square and / or modulated and / or modulated square wave waveforms of variable capacitance and voltage. Contains one or more pulses. References to electric and electrical fields must be construed as including references to the presence of potential differences in the cellular environment. Such an environment can be set up by static electricity, alternating current (AC), direct current (DC), etc., as is known in the art. The electric field may be uniform, non-uniform or otherwise, and the intensity and / or direction may change in a time-dependent manner.

A single or multiple application of an electric field and a single or multiple application of ultrasonic waves are also possible in any order and in any combination. Ultrasound and / or electric fields may be delivered as single or multiple continuous applications, or as pulses (pulse delivery).

Electroporation has been used in both in vitro and in vivo procedures for introducing exogenous material into living cells. For in vitro application, a live cell sample is first mixed with the drug of interest and placed between electrodes such as parallel plates. The electrodes then apply an electric field to the cell / implant mixture. Examples of systems for performing in vitro electroporation include Electro Cell Manipulator ECM600 products, all manufactured by Genetronics, Inc.'s BTX Division, and Electro Square Portor T820 (US Pat. No. 5,869,326). reference).

Known electroporation methods (both in vitro and in vivo) work by applying short high voltage pulses to electrodes positioned around the treatment area. The electric field generated between the electrodes temporarily makes the cell membrane porous, and then the molecules of the drug of interest invade the cell. In known electroporation applications, this electric field contains a single square wave pulse of about 1000 V / cm with a duration of about 100 μs. Such pulses can be generated, for example, in known applications of the Electro Square Portor T820.

Preferably, the electric field has an intensity of about 1 V / cm to about 10 kV / cm under in vitro conditions. Therefore, the electric field is 1V / cm, 2V / cm, 3V / cm, 4V / cm, 5V / cm, 6V / cm, 7V / cm, 8V / cm, 9V / cm, 10V / cm, 20V / cm, 50V /. cm, 100V / cm, 200V / cm, 300V / cm, 400V / cm, 500V / cm, 600V / cm, 700V / cm, 800V / cm, 900V / cm, 1kV / cm, 2kV / cm, 5kV / cm, It can have an intensity of 10 kV / cm, 20 kV / cm, 50 kV / cm or more. More preferably, it is about 0.5 kV / cm to about 4.0 kV / cm under in vitro conditions. Preferably the electric field has an intensity of about 1 V / cm to about 10 kV / cm under in vivo conditions. However, the electric field strength may be low if the number of pulses delivered to the target site is increased. Therefore, pulsed electric field delivery with lower electric field strength is envisioned.

Preferably, the application of the electric field is in the form of multiple pulses, such as double pulses of the same intensity and capacitance, or sequential pulses of varying intensity and / or capacitance. As used herein, the term "pulse" includes one or more waveforms of variable capacitance and voltage, including exponential and / or square and / or modulated or modulated square wave waveforms. Includes electrical pulses.

Preferably, the electrical pulse is delivered as a waveform selected from an exponential waveform, a square waveform, a modulated waveform and a modulated square waveform.

In certain preferred embodiments, a low voltage direct current is used. Therefore, Applicants disclose the use of an electric field applied to a cell, tissue or tissue mass at an electric field strength of 1 V / cm to 20 V / cm for a time of 100 ms or longer, preferably 15 minutes or longer.

Ultrasound is preferably administered at a power level of about 0.05 W / cm ² to about 100 W / cm ² . Diagnostic or therapeutic ultrasound, or a combination thereof, may be used.

As used herein, the term "ultrasound" refers to energy in the form of mechanical vibrations whose frequencies are so high that they exceed the human auditory range. The lower limit frequency of the ultrasonic spectrum can generally be considered to be about 20 kHz. Frequencies in the 1-15 MHz range are used in many diagnostic applications of ultrasound (Source: Ultrasonics in Clinical Diagnostics, PNT Wells, ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, New York] , 1977]).

Ultrasound has been used for both diagnostic and therapeutic applications. When used as a diagnostic tool (“diagnostic ultrasound”), ultrasound is typically used in an energy density range of up to about 100 mW / cm ² (FDA recommended), but with an energy density of up to 750 mW / cm ^2. It is used. In physiotherapy, ultrasound is typically used as an energy source in the range of up to about 3-4 W / cm ² (WHO recommended). In other therapeutic applications, higher intensity ultrasound may be utilized, for example, in HIFU a short time of 100 W / cmup to 1 kW / cm ² (or even higher). The term "ultrasound", as used herein, is intended to include diagnostic, therapeutic and focused ultrasound.

Focused ultrasound (FUS) enables the delivery of thermal energy without invasive probes (see Morocz et al 1998 Journal of Journal Imaging Vol. 8, No. 1, pp. 136-142. The focused ultrasound of the form is a high density focused ultrasound (HIFU), which is Moussatov et al in Ultrasonics (1998) Vol. 36, No. 8, pp. 893-900 and Tran Huu Hue et al in Acoustica (1997) ) Vol.83, No.6, pp.1103-1106.

Preferably, a combination of diagnostic ultrasound and therapeutic ultrasound is utilized. However, this combination is not intended to be limited and one of ordinary skill in the art will appreciate that any variety of combinations of ultrasound can be used. In addition, energy density, ultrasonic frequency, and exposure time can vary.

Preferably the exposure to the ultrasonic energy source is a power density of about 0.05 to about 100 Wcm- ² . Even more preferably, exposure to an ultrasonic energy source is a power density of about 1 to about 15 Wcm- ² .

Preferably the exposure to the ultrasonic energy source is at a frequency of about 0.015-about 10.0 MHz. More preferably, exposure to an ultrasonic energy source is at a frequency of about 0.02 to about 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasonic waves are applied at a frequency of 3 MHz.

Preferably the exposure is from about 10 ms to about 60 minutes. Preferably the exposure is from about 1 second to about 5 minutes. More preferably, the ultrasonic waves are applied for about 2 minutes. However, the exposure may be for a longer duration, eg 15 minutes, depending on the detailed target cells to be destroyed.

Advantageously, the target tissue is exposed to ultrasonic energy source frequency at about 0.05Wcm ^-2 ~ about acoustic power density of 10Wcm ^-2 in the range of about 0.015 to about 10 MHz (WO 98 / See Pamphlet No. 52609). However, it is also possible to expose to an ultrasonic energy source in another way, eg, at a sound power density greater than 100 Wcm- ² , but for a shorter period of time, eg 1000 Wcm- ² , for less than a millisecond range.

Preferably the application of ultrasonic waves is in the form of multiple pulses; therefore, both continuous and pulsed waves (pulse ultrasonic delivery) can be utilized in any combination. For example, continuous wave ultrasound may be applied followed by pulsed wave ultrasound, and vice versa. This may be repeated any number of times, in any order and in any combination. A pulsed wave ultrasound may be applied to the background of the continuous wave ultrasound and any pulse can be used in any cohesiveness.

Preferably, the ultrasound may include pulsed ultrasound. In a highly preferred embodiment, the ultrasonic waves are applied as continuous waves with a power density of 0.7 Wcm- ² or 1.25 Wcm- ² . Higher power densities may be used when pulsed ultrasound is used.

The use of ultrasound, like light, is advantageous because it can be accurately focused on the target. Furthermore, ultrasonic waves, unlike light, are advantageous because they can be focused deeper in the tissue. Ultrasound is therefore better suited for whole tissue permeation therapy (such as, but not limited to, liver lobe) or whole organ therapy (such as, but not limited to, whole liver or muscle, such as the heart). Another important advantage is that ultrasound is a non-invasive stimulus used in a wide variety of diagnostic and therapeutic applications. As an example, ultrasound is well known in medical imaging techniques, as well as in orthopedic therapy. In addition, devices suitable for applying ultrasonic waves to target vertebrates are widely available and their use is well known in the art.

The rapid transcriptional response and endogenous targeting of the present invention create an ideal system for transcriptional dynamics studies. For example, the present invention can be used to study the dynamics of mutant formation during inducible expression of a target gene. On the other side of the transcription cycle, mRNA degradation studies that cause changes in expression levels of large numbers of genes in response to strong extracellular stimuli are often performed. The present invention can be used to reversibly induce transcription of an endogenous target, stop stimulation after the time of transcription, and track the rate of degradation of a unique target.

The temporal accuracy of the present invention may provide the ability to adjust the timing of gene regulation in response to experimental intervention. For example, targets suspected of being involved in long-term potentiation (LTP) are in organ-type or dissociated nerve cultures, but only while the stimulus induces LTP to prevent disruption of normal cell development. Can be adjusted. Similarly, in a cell model exhibiting a disease phenotype, targets suspected of being involved in the effectiveness of a particular treatment can be regulated only during treatment. Conversely, genetic targets can only be regulated during the presence of pathological stimuli. Any experiment in which the timing of the genetic cue to an external experimental stimulus makes sense can potentially benefit from the usefulness of the present invention.

Similarly, in the in vivo context, there are many opportunities for the present invention to control gene expression. Light inducibility offers the possibility of spatial accuracy. Utilizing the development of optoload technology, stimulating fiber optic leads can be placed in the correct brain region. The stimulation region size can then be adjusted by light intensity. This may be done in conjunction with the delivery of the Cas9 CRISPR-Cas system or complex of the invention, or in the case of transgenic Cas9 animals, the guide RNA of the invention may be delivered by optrol technology. It may be possible to regulate gene expression in accurate brain regions. A clear Cas9-expressing organism can have a guide RNA of the invention administered to it, and then there can be highly accurate changes in laser-induced local gene expression.

Culture media for culturing host cells include, in particular, M199 Earl, Eagle's MEM (E-MEM), Dalbeco MEM (DMEM), SC-UCM102, UP-SFM (GIBCO BRL), EX-CELL302 (Nichirei). , EX-CELL293-S (Nichirei), TFBM-01 (Nichirei), ASF104, and other media commonly used for tissue culture. Suitable culture media for a particular cell type can be found in the American Type Culture Collection (ATCC) or the European Collection of Cell Cultures (ECACC). it can. The culture medium may be supplemented with amino acids such as L-glutamine, salts, antifungal or antibacterial agents such as Fungizone®, penicillin-streptomycin, animal serum and the like. The cell culture medium may be optionally serum-free.

The present invention may also provide valuable temporal accuracy in vivo. The present invention can be used to alter gene expression during a particular developmental stage. The present invention can be used to adjust the timing of genetic queuing for a particular experimental window. For example, genes involved in learning can be overexpressed or suppressed in the exact region of the intact rodent or primate brain as long as there is a learning stimulus. Furthermore, the present invention can be used to induce changes in gene expression only during a particular disease onset stage. For example, an oncogene can be overexpressed only when the tumor reaches a certain size or stage of metastasis. Conversely, proteins suspected of developing Alzheimer's can be knocked down only at defined points in the life of the animal and within specific brain regions. Although these examples do not exhaustively list the possible applications of the present invention, they do reveal some areas in which the present invention can be a powerful technique.

Protected guide: The enzyme according to the present invention can be used in combination with a protected guide RNA. In one embodiment, the object of the present invention is to determine the specificity of an individual guide RNA conferred on Cas9 and the target DNA of the guide RNA. It is further enhanced by thermodynamic adjustment of binding specificity to. It introduces a guide sequence mismatch, elongation or truncation so that the targeted genomic locus has a thermodynamic advantage over genomic off-target, so that the genomic target and its potential off-target locus are It is a general technique for increasing / decreasing the number of complementary base pairs and mismatched bases shared with.

In one aspect, the invention provides a guide sequence modified by secondary structure to increase the specificity of the Cas9 CRISPR-Cas system, thereby protecting the secondary structure from exonuclease activity and the guide sequence. It may be possible to add 3'to.

In one aspect, the invention provides to hybridize a "protective RNA" to a guide sequence, where the "protective RNA" is complementary to the 5'end of the guide RNA (gRNA), thereby partially. It is an RNA strand that produces a positive double-stranded gRNA. In certain embodiments of the invention, protecting the mismatched base with a fully complementary protective sequence reduces the likelihood that the target DNA will bind to the mismatched base pair at the 3'end. In embodiments of the invention, additional sequences may also be present, including extended lengths.

Guide RNA (gRNA) extensions that match genomic targets provide gRNA protection and enhance specificity. Elongation of gRNA by matching sequences distal to the end of the spacer seed for individual genomic targets is envisioned to result in increased specificity. Matched gRNA elongations that enhance specificity have been observed in cells without truncation. Predictions of gRNA structures with these stable length extensions indicate that the protected state results in a stable morphology, where the extension is due to the complementary sequence of the spacer extension and the spacer seed. To form a closed loop with the gRNA seed. These results demonstrate that the concept of protected guides also includes sequences that match genomic target sequences distal to the 20mer spacer binding region. Thermodynamic predictions can be used to predict perfectly matched or partially matched guide extensions that result in a protected gRNA state. This extends the concept of protected gRNAs to the interaction between X and Z, where X is generally 17-20 nt in length and Z is 1-30 nt in length. Thermodynamic conjectures can be used to determine the optimal elongation state of Z, which potentially introduces a small number of mismatches into Z and promotes the formation of a protected conformation between X and Z. .. Throughout the application, the terms "X" and seed length (SL) are used synonymously with the term exposure length (EpL), which means the number of nucleotides available for binding the target DNA; the term " "Y" and protection length (PL) are used synonymously to represent the length of the protector; and the terms "Z", "E", "E'" and EL are nucleotides that extend the target sequence. It is used synonymously with the extension length (ExL) of the term representing the number of.

The extension sequence corresponding to the extension length (ExL) may optionally be added directly to the guide sequence at the 3'end of the protected guide sequence. The extension sequence may be 2-12 nucleotides in length. Preferably ExL is represented as 0, 2, 4, 6, 8, 10 or 12 nucleotides in length. In a preferred embodiment, ExL is represented as 0 or 4 nucleotides in length. In a more preferred embodiment, ExL is 4 nucleotides in length. The extension sequence may or may not be complementary to the target sequence.

The extension sequence may further optionally be added directly to the guide sequence at the 5'end of the protected guide sequence and directly to the 3'end of the protected sequence. As a result, the extension sequence acts as a linking sequence between the protected sequence and the protected sequence. Although not desired to be constrained by theory, the binding of the protected sequence to the protected sequence can be improved because the sequence protected by such concatenation is placed near the protected sequence.

Addition of a gRNA mismatch to the distal end of the gRNA may indicate enhanced specificity. Introducing an unprotected distal mismatch in Y, or extending a gRNA with a distal mismatch (Z), may indicate enhanced specificity. This concept, as described, relates to the X, Y, and Z components used in protected gRNAs. The concept of unprotected mismatch can be further generalized to the concept of X, Y, and Z described for protected guide RNAs.

In one aspect of Cas9 Cas9, the invention provides enhanced Cas9 Cas9 specificity, where two results can be considered from the double-stranded 3'end of a protected guide RNA (pgRNA): (1) Guide RNA. -A strand exchange from the protected RNA to the guide RNA-the target DNA occurs and the guide binds completely to the target, or (2) the guide RNA cannot bind completely to the target and Cas9 target cleavage occurs in the Cas9 catalytic DSB. Guide RNA for activation: Cas9 cleavage does not occur if the guide RNA does not bind properly here because it is a multi-step kinetic reaction that requires target DNA binding. According to detailed embodiments, protected guide RNAs improve the specificity of target binding when compared to the naturally occurring CRISPR-Cas system. According to detailed embodiments, protected modified guide RNAs improve stability when compared to naturally occurring CRISPR-Cas. According to a detailed embodiment, the protected sequence is 3 to 120 nucleotides in length and comprises three or more contiguous nucleotides complementary to another guide sequence or protected sequence. According to a detailed embodiment, the protective sequence forms a hairpin. According to a detailed embodiment, the guide RNA further comprises a protected sequence and an exposed sequence. According to a detailed embodiment, the exposed sequence is 1-19 nucleotides. More specifically, the exposed sequence is at least 75%, at least 90% or about 100% complementary to the target sequence. According to a detailed embodiment, the guide sequence is at least 90% or about 100% complementary to the protective strand. According to a detailed embodiment, the guide sequence is at least 75%, at least 90% or about 100% complementary to the target sequence. According to a detailed embodiment, the guide RNA further comprises an extension sequence. More specifically, the extension sequence is operably linked to the 3'end of the protected guide sequence and optionally directly to the 3'end of the protected guide sequence. According to a detailed embodiment, the extension sequence is 1-12 nucleotides. According to a detailed embodiment, the extension sequence is operably linked to the guide sequence at the 3'end of the protected guide sequence and to the 5'end of the protective chain, optionally to the 3'end of the protected guide sequence and protection. Directly linked to the 3'end of the strand, where the extension sequence is the linking sequence between the protected sequence and the protected strand. According to a detailed embodiment, the extension sequence is 100% non-complementary to the protected strand and optionally at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least the protected strand. It is 50% non-complementary. According to a detailed embodiment, the guide sequence further comprises a mismatch added to the end of the guide sequence, and these mismatches thermodynamically optimize the specificity.

In one aspect, the invention provides an engineered non-naturally occurring CRISPR-Cas system comprising a Cas9 protein and a protected guide RNA that targets a DNA molecule encoding a gene product in a cell, thereby providing a non-natural CRISPR-Cas system. A protected guide RNA targets the DNA molecule that encodes the gene product, and the Cas9 protein cleaves the DNA molecule that encodes the gene product, thereby altering the expression of the gene product; and here with the Cas9 protein. Protected guide RNA does not exist together in nature. The present invention includes a protected guide RNA containing a guide sequence in which 3'is fused to a direct repeat sequence. The present invention further includes Cas9 proteins that are codon-optimized for expression in eukaryotic cells. In a preferred embodiment, the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced. In some embodiments, the Cas9 enzyme is a Cas9 derived from the genus Acidaminococcus sp. BV3L6, a Lachnospiraceae bacterium or a Francisella novicida organism derived from Francisella novicida. It may include Cas9. The enzyme may be an additional Cas9 homolog or ortholog. In some embodiments, the nucleotide sequence encoding the Cfp1 enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the Cas9 enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In general, and throughout the specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; free-ended (eg, circular) nucleic acid molecules containing one or more free ends; DNA, Nucleic acid molecules containing RNA, or both; and other types of polynucleotides known in the art. Certain vectors are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where the vector is a virus for packaging into viruses (eg, retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus). The derived DNA or RNA sequence is present. Viral vectors also include polynucleotides carried by the virus for transfection into host cells. A particular vector is capable of self-renewal in the host cell into which it is introduced (eg, a bacterial vector with a bacterial origin of replication and an episomal mammalian vector). Other vectors (eg, non-episome mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. In addition, a particular vector can guide the expression of a gene to which it is operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

The recombinant expression vector can contain a form of nucleic acid of the invention suitable for expression of the nucleic acid in the host cell, that is, one in which the recombinant expression vector is operably linked to the nucleic acid sequence to be expressed. It is meant to include the above regulatory elements, which may be selected based on the host cell used for expression. Within the scope of the recombinant expression vector, "operably linked" allows expression of the nucleotide sequence (eg, in an in vitro transcription / translation system, or in the host cell when the vector is introduced into the host cell). It is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements.

Advantageous vectors include lentivirus and adeno-associated virus, and the type of such vector can also be selected for targeting specific types of cells.

In one aspect, the invention is a first operably linked to (a) a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences downstream of the direct repeat sequence. Regulatory elements (guide sequences, when expressed, lead to sequence-specific binding of the CRISPR complex to the target sequence in eukaryotic cells, and the CRISPR complex is a complex with a guide RNA containing a guide sequence that hybridizes to the target sequence. Eukaryotic host cells containing a second regulatory element operably linked to the enzyme coding sequence encoding the Cas9 enzyme (including the CRISPR enzyme that formed) and / or (b) the nuclear localization sequence. provide. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, the components (a), components (b), or components (a) and (b) are stably integrated into the genome of the host eukaryotic cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it leads to sequence-specific binding of the CRISPR complex to different target sequences in eukaryotic cells. In some embodiments, the Cas9 enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the Cas9 enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter.

In some embodiments, the invention provides a non-human eukaryote, preferably a multicellular eukaryote, comprising a eukaryotic host cell according to any of the described embodiments. In another aspect, the invention provides a eukaryote comprising a eukaryotic host cell according to any of the described embodiments; preferably a multicellular eukaryote. The organism in some embodiments of these embodiments may be an animal; eg, a mammal. In addition, the organism may be an arthropod such as an insect. The organism may also be a plant or yeast. In addition, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more of the constituents described above herein. In some embodiments, the kit includes a vector system and instructions for use of the kit. In some embodiments, the vector system is operably linked to (a) a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences downstream of the direct repeat sequence. The first regulatory element (the guide sequence, when expressed, leads to sequence-specific binding of the Cas9 CRISPR complex to the target sequence in eukaryotic cells, and the CRISPR complex contains a protection that contains a guide sequence that hybridizes to the target sequence. Includes a second regulatory element operably linked to an enzyme coding sequence encoding the Cas9 enzyme (including a Cas9 enzyme complexed with a type guide RNA) and / or (b) a nuclear localization sequence. .. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it leads to sequence-specific binding of the CRISPR complex to different target sequences in eukaryotic cells. In some embodiments, the Cas9 enzyme comprises one or more nuclear localization sequences that are strong enough to drive the accumulation of a detectable amount of the Cas9 enzyme in the nucleus of a eukaryotic cell. In some embodiments, the Cas9 enzyme is found in the genus Acidaminococcus sp. BV3L6, the Lachnospiraceae bacterium MA2020, or the Francisella tularensis 1 in Francisella tularensis, CRISPR, and CRISPR. It may include the derived mutant Cas9. The enzyme may be Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter.

In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding the CRISPR complex to a target polynucleotide causing cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex is: , Includes a Cas9 enzyme complexed with a protected guide RNA containing a guide sequence that hybridizes to the target sequence within the target polynucleotide. In some embodiments, the cleavage comprises cleaving one or two strands at the position of the target sequence with the Cas9 enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises the step of repairing the cleaved target polynucleotide by a non-homologous end joining (NHEJ) based gene insertion mechanism, more specifically with an exogenous template polynucleotide. The repair results in a mutation involving the insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in the protein expressed from the gene containing the target sequence. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, where the one or more vectors are a guide sequence linked to a Cas9 enzyme, a direct repeat sequence. Drives the expression of one or more of the protected guide RNAs, including. In some embodiments, the vector is delivered to a subject eukaryotic cell. In some embodiments, the modification occurs in the eukaryotic cell in a cell culture. In some embodiments, the method further comprises the step of isolating the eukaryotic cell from the subject prior to the modification. In some embodiments, the method further comprises the step of returning the eukaryotic cell and / or cells derived thereto to the subject.

In one aspect, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding the Cas9 CRISPR complex to a polynucleotide, which causes an increase or decrease in the expression of the polynucleotide; where the CRISPR complex is described above. It contains a Cas9 enzyme complexed with a protected guide RNA containing a guide sequence that hybridizes to a target sequence within a polynucleotide. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, wherein the one or more vectors are one of Cas9 enzymes and protected guide RNAs. Drive the above expression.

In one aspect, the invention provides a method of creating a model eukaryotic cell containing a mutant disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method is: (a) a step of introducing one or more vectors into eukaryotic cells (one or more vectors are protected containing a Cas9 enzyme and a guide sequence linked to a direct repeat sequence. Drives the expression of one or more of the type guide RNAs); and (b) the step of binding the CRISPR complex to a target polynucleotide causing cleavage of the target polynucleotide within the disease gene (the CRISPR complex is , Containing a Cas9 enzyme complexed with a guide RNA containing a sequence that hybridizes to a target sequence within a target polynucleotide) thereby creating a model eukaryotic cell containing a mutant disease gene. In some embodiments, the cleavage comprises cleaving one or two strands at the position of the target sequence with the Cas9 enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing the cleaved target polynucleotide with an exogenous template polynucleotide by a non-homologous end joining (NHEJ) based gene insertion mechanism, wherein the repair comprises said. It results in a mutation involving the insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in protein expression from the gene containing the target sequence.

In one aspect, the invention provides a method of developing a biologically active agent that regulates cell signaling events associated with a disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method involves contacting the test compound with a model cell of any one of the embodiments described; and (b) a cell signal associated with the mutation of the disease gene. The biologically active agent is developed that comprises detecting changes in readings that are indicators of decreased or increased transduction events, thereby regulating said cell signaling events associated with said disease gene.

In one aspect, the invention provides a recombinant polynucleotide comprising a protected guide sequence downstream of a direct repeat sequence, wherein the protected guide sequence, upon expression, to a corresponding target sequence present in eukaryotic cells. Leads to sequence-specific binding of the CRISPR complex. In some embodiments, the target sequence is a viral sequence that is present in eukaryotic cells. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

In one aspect, the invention provides a method of selecting one or more cells by introducing one or more mutations into one or more intracellular genes, the method of which is one or more. Steps to introduce one or more vectors into cells (one or more vectors drive expression of one or more of Cas9 enzymes, protected guide RNAs containing guide sequences, and editing templates; edited here The template contains one or more mutations that abolish Cas9 enzyme cleavage); non-homologous end joining (NHEJ) -based gene insertion of the editing template at a target polynucleotide in one or more cells of choice. Steps to enable the mechanism; steps to bind the CRISPR complex to the target polynucleotide and cause cleavage of the target polynucleotide in the gene (the CRISPR complex is a guide sequence that hybridizes to the target sequence within the target polynucleotide). Includes a Cas9 enzyme complexed with a protected guide RNA containing, where CRISPR complex binding to a target polynucleotide induces cell death), thereby introducing one or more mutations. Allows selection of one or more cells. In a preferred embodiment of the invention, the cell of choice may be a eukaryotic cell. Aspects of the invention allow the selection of specific cells that do not require a two-step process that may include selectable markers or counterselective systems.

With respect to mutations in the Cas9 enzyme, if the enzyme is not FnCas9, the mutation may be as described elsewhere in this specification; conservative substitutions of any of the substituted amino acids are also envisioned. In some embodiments, the invention provides with respect to any or each or all embodiments discussed herein, wherein the CRISPR enzyme comprises at least one or more, or at least two or more mutations. Here, at least one or more mutations or at least two or more mutations are selected from those described elsewhere herein.

In a further aspect, the invention binds to a computer-assisted method for identifying or designing a potential compound to fit in or bind to a part of the CRISPR-Cas9 system or its functionality or vice versa (binding to a desired compound). As related to predicting the operable range of the potential CRISPR-Cas9 system or a potential CRISPR-Cas9 system (eg, a CRISPR-Cas9 system) or a computer-assisted method for identifying or designing a portion of its functionality. -For example, based on crystal structure data or based on Cas9 ortholog data, or in relation to where functional groups such as activators or repressors can be added to the CRISPR-Cas9 system, or with respect to Cas9 truncation or Includes computer-assisted methods for identifying or designing (with respect to nickase design), said methods.
Using a computer system, eg, a programmed computer including a processor, a data storage system, an input device, and an output device, the following steps:
(A) For example, three-dimensional coordinates of a subset of atoms from or related to the CRISPR-Cas9 crystal structure in different domains based on differences between Cas9 orthologs in, or in place of, or in addition to, the CRISPR-Cas9 system binding domain. Data containing, or with respect to Cas9, nickase, or functional groups, is optionally entered into a computer programmed using the input device, along with structural information from one or more CRISPR-Cas9-based complexes. , Thereby creating a dataset;
(B) A computer database of structures stored in the computer data storage system using the processor, eg, structures of compounds that bind or presumably bind to the CRISPR-Cas9 system or compounds that are desirable to bind to it. And, or with respect to Cas9 orthologs (eg, as Cas9 or with respect to different domains or regions between Cas9 orthologs) or with respect to CRISPR-Cas9 crystal structures or with respect to nickases or with respect to functional groups;
(C) Using a computer method, one or more structures-eg, a CRISPR-Cas9 structure capable of binding to a desired structure, a desired structure capable of binding to a particular CRISPR-Cas9 structure, a CRISPR-Cas9 crystal structure. A part of the CRISPR-Cas9 system that can be manipulated, eg, based on data from other parts and / or data from the Cas9 ortholog, a truncated Cas9, a novel nickase or a specific functional group, or a position or function to add a functional group. Steps to select the sex group-CRISPR-Cas9 system from the database;
(D) A step of constructing a model of one or more selected structures using a computer method; and (e) A step of outputting the selected one or more structures to the output device;
And optionally, the step of synthesizing one or more of the selected one or more structures;
And, optionally, including the step of testing one or more structures of the synthesized selection as or in the CRISPR-Cas9 system.
Alternatively, the method comprises the coordinates of at least two atoms of the CRISPR-Cas9 crystal structure, eg, the coordinates of at least two atoms in the CRISPR-Cas9 crystal structure table herein or at least a subdomain of the CRISPR-Cas9 crystal structure. Steps that provide coordinates (“selection coordinates”), CRISPR-that can be manipulated based on data from candidate structures containing binding molecules or, for example, data from other parts of the CRISPR-Cas9 crystal structure and / or data from the Cas9 ortholog. A step that provides the structure of a portion of the Cas9 system, or the structure of a functional group, and a CRISPR-Cas9 structure, a specific CRISPR-Cas9 structure that can fit a candidate structure to selected coordinates and thereby bind to the desired structure. A desired structure that can be attached to, a portion of the CRISPR-Cas9 system that can be manipulated, a truncated Cas9, a novel nickase, or a specific functional group, or a position or functional group to which a functional group is added-the CRISPR-Cas9 system. The step of obtaining the including product data with its output; and optionally, including the step of synthesizing one or more compounds from the product data, and optionally, the synthesized one or more. Includes steps to test the compounds of CRISPR-Cas9 as or in the CRISPR-Cas9 system.

The steps tested may include analyzing the CRISPR-Cas9 system obtained from one or more of the synthesized selection structures, eg, with respect to binding or with respect to performing the desired function.

The output of the aforementioned method includes documents such as data transmission, eg telecommunications, telephone, video conferencing, the media, eg presentations such as computer presentations (eg PowerPoint), the Internet, email, computer program (eg WORD) documents. It may include the transmission of information by communication by. Therefore, the present invention is also atomic coordinate data based on the crystal structure referred to herein, which defines the three-dimensional structure of CRISPR-Cas9 or at least one subdomain thereof, or structural factors of CRISPR-Cas9. The data also includes computer-readable media containing structural factor data that can be derived from the atomic coordinate data of the crystal structure referred to herein. The computer-readable medium may also contain any data from the methods described above. The present invention further relates to methods, atomic coordinate data based on the crystal structure referenced herein, data defining the three-dimensional structure of CRISPR-Cas9 or at least one subdomain thereof, or structural factors of CRISPR-Cas9. Includes computer systems for generating or implementing rational designs as described above, including data that include any of the structural factor data that can be derived from the atomic coordinate data of the crystal structure referenced herein. To do. The present invention further relates to a method of conducting business, a three-dimensional structure of a computer system or medium or CRISPR-Cas9 or at least one subdomain thereof, or structural factor data of CRISPR-Cas9, which is referred to herein. A method comprising providing the user with the structure shown in the atomic coordinate data of the crystal structure to be obtained and the structural factor data derived from the structure, or the computer medium in the present specification or the data transmission in the present specification.

A "binding site" or "active site" is a site within a binding cavity or region (such as an atom, a functional group of amino acid residues or multiple such atoms and / or groups) that binds to a compound such as a nucleic acid molecule. Possible, includes, or consists essentially of, a site involved in binding.

"Fiting" is the determination of the interaction between one or more atoms of a candidate molecule and at least one atom of the structure of the invention by automatic or semi-automatic means, and to what extent such interaction is stable. It means to calculate what you are doing. Interactions include attractive and repulsive forces brought about by electric charges, three-dimensional considerations, and the like. Various computer-based fitting methods are further described.

By "root mean square (or rms) deviation", we mean the square root of the arithmetic mean of the square of the deviation from the mean.

"Computer system" means hardware means, software means and data storage means used for analysis of atomic coordinate data. Minimal hardware means for computer-based systems of the present invention typically include a central processing unit (CPU), input means, output means and data storage means. Desirably, a display or monitor is provided to visualize the structural data. The data storage means may be RAM or a means of accessing the computer-readable medium of the present invention. Examples of such systems are computers and tablet devices running Unix, Windows or Apple operating systems.

"Computer-readable medium" means any one or more media that can be read and accessed directly or indirectly by a computer, eg, to be a medium suitable for use in the computer systems described above. Such media are not limited, but are limited to magnetic storage media such as floppy disks, hard disk storage media and magnetic tapes; optical storage media such as optical disks or CD-ROMs; electrical storage media such as RAM and ROM; thumb drive devices; cloud Hybrids in these categories such as storage devices and magnetic / optical storage media.

The present invention includes the use of the protected guides described above herein in the optimized functional CRISPR-Cas enzyme system described herein.

Forming RISC by Guide Engineering In some embodiments, the guide may be a protected guide (eg, pgRNA) or an escorted guide (eg, esgRNA) as described herein. Both of these utilize RISC in some embodiments. RISC is a major component of RNAi. RISC (RNA-induced silencing complex) is a double-stranded RNA (siRNA) or microRNA (miRNA) that acts as a template for RISC and recognizes complementary messenger RNA (mRNA) transcripts. A multiprotein complex that incorporates one strand of a dsRNA) fragment, specifically a ribonuclear protein complex. Therefore, mRNA is cleaved by one of the constituents of RISC.

Therefore, the formation of RISC is advantageous in some embodiments. Guide RNAs according to various aspects of the invention include, but are not limited to, protected and / or escorted guide RNAs, eg, siRNAs that may be provided to cells or may, for example, already be expressed in cells. Alternatively, in combination with miRNA, it may be adapted to include RNA nucleotides that promote the formation of RISCs. This can be useful for removing or degrading guides, for example as a self-inactivating system.

Thus, the guide RNA may contain sequences complementary to the target miRNA or siRNA that may or may not be present in the cell. In this way, there is binding of the RNA sequence to the miRNA or siRNA only when the miRNA or siRNA is present, for example, via expression (either by cells or through human intervention), which in turn becomes intracellular. RNA-induced silencing complex (RISC) results in cleavage of guided RNA. Thus, in some embodiments, the guide RNA comprises an RNA sequence complementary to the target miRNA or siRNA, and when the guide RNA sequence binds to the target miRNA or siRNA, an RNA-induced silencing complex within the cell ( It results in cleavage of the guide RNA by RISC).

This will be further described below with specific reference to both protected guides and escorted guides.

CRISPR formation with protected guides For example, protected guides can be described in the following embodiments: (a) protected sequences, (b) guide sequences capable of hybridizing to target sequences in eukaryotic cells, (c). ) Tracr mate sequence, and (d) a clustered, regularly spaced short-interval Palindrome repeat (CRISPR) -CRISPR-related (cas) (CRISPR-Cas) with a protected guide RNA (pgRNA) polynucleotide sequence containing the tracr sequence. ) Systems, engineered non-naturally occurring compositions [(a), (b), (c) and (d) are aligned from 5'to 3', and the protected sequence is non-complementary to the target sequence. When transcribed with two or more nucleotides, the tracr mate sequence hybridizes to the tracr sequence, the guide sequence leads to sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex is (1). It contains a type II Cas9 protein complexed with 1) a guide sequence that hybridizes to the target sequence, and (2) a tracr mate sequence that hybridizes to the tracr sequence, and in the polynucleotide sequence, the guide, tracr and tracr mate. One or more of the sequences have been modified].

In one aspect, this protected guide system is used to protect the secondary structure of the 5'extension to the sgRNA. For example, Applicants extend an sgRNA such that a miRNA binding site is introduced to activate the sgRNA only when the miRNA binding site is processed and cleaved by the RISC complex mechanism. This is not possible without secondary structure protection, as exonuclease processing begins at the 5'end and cleaves back towards the sgRNA. By adding a small secondary structure loop to the 5'side of the added miRNA site, the miRNA can be protected from chewing back of the exonuclease.

RISC formation with escorted guides In another example, escorted guides can be described. In particular, miRNA-induced esgRNA is envisioned. Here, the escort RNA aptamer sequence is complementary to the target miRNA, so when the target miRNA is present in cells incorporated into RNA-induced silencing complex (RISC), the binding of the escort RNA aptamer sequence to the target miRNA Yes, which results in cleavage of esgRNA by RNA-induced silencing complex (RISC) in the cell.

In an alternative embodiment, the 5'end of the esgRNA may be provided with a wide variety of primary and secondary structures designed so that the RISC complex has access to the miRNA binding site. The esgRNA may have first and second linker sequences on the 5'side of the protected sequence. In an alternative embodiment, the linkers 1 and 2 may be, for example, independently 0, 1, 2, 3 or 4 nucleotides in length, respectively, where the protected sequence is 0, 1 or 2 nucleotides in length.

In an exemplary embodiment, the induction of esgRNA targeting is such that miR-122 is not naturally expressed in HEK. It can be illustrated using miR-122 in a 293 cell line. In the absence of exogenous miR-122, protected esgRNA did not mediate target EMX1.3 nuclease activity. When exogenous miR-122 was added (100 ng / well), target EMX1.3 cleavage was observed (as a characteristic cleavage artifact visible as an electrophoretic variant on the gel). This demonstrates that highly expressed endogenous miRNAs can be utilized in systems that provide genetically inducible sgRNAs. Any miRNA can be used in place of the miRNA 122 and the corresponding sequence is readily determined.

For example, the sgRNA may be linked to an "escort" RNA aptamer sequence complementary to the endogenous target miRNA. Target miRNAs can form RNA-induced silencing complexes (RISCs) in cells. When the target miRNA is present in the cell, there is binding of the escort RNA aptamer sequence to the target miRNA, which results in cleavage of the esgRNA by RNA-induced silencing complex (RISC) in the cell. Cleavage of the escort releases the active sgRNA.

For example, a protected guide can be described in the following ways:
An RNA-guided sequence that can hybridize to a target sequence at the target genomic locus of the cell;
Escort RNA aptamer sequences [Escort RNA aptamer sequences contain binding affinity for intracellular or intracellular aptamer ligands, or escort RNA aptamer sequences are responsive to intracellular or intracellular localized aptamer effectors.
The presence of aptamer ligands or effectors on or within cells is spatially or temporally restricted] in naturally occurring or engineered compositions containing single CRISPR-Cas9 guide RNA (esgRNA) with escorts] ..

The escort RNA aptamer sequence may be complementary to the target miRNA that may or may not be present in the cell, thereby binding the escort RNA aptamer sequence to the target miRNA only in the presence of the target miRNA. This results in the cleavage of esgRNA by RNA-induced silencing complex (RISC) in the cell. Thus, in some embodiments, the escort RNA aptamer sequence is complementary to the target miRNA, and when the escort RNA aptamer sequence binds to the target miRNA, the intracellular RNA-induced silencing complex (RISC) of the esgRNA A disconnect is brought about.

According to the invention, the nucleotide sequence encoding at least one of the guide RNA or Cas protein is operably linked intracellularly to a regulatory element containing the promoter of the gene of interest, thereby at least one CRISPR-. Expression of Cas-based components is driven by the promoter of the gene of interest. "Operabably connected" is one in which the nucleotide sequence encoding the guide RNA and / or Cas allows the expression of the nucleotide sequence, as is similarly referred to elsewhere herein. Or intended to mean connected to multiple adjustment elements. The term "adjustment element" is also described elsewhere herein. According to the present invention, the regulatory element preferably comprises a promoter of the gene of interest, such as a promoter of the endogenous gene of interest. In certain embodiments, the promoter is at its endogenous genomic position. In such embodiments, the nucleic acid encoding CRISPR and / or Cas is under transcriptional control of the promoter of the gene of interest at its native genomic position. In certain other embodiments, the promoter is provided on (separate) nucleic acid molecules such as vectors or plasmids, or on other extrachromosomal nucleic acids, i.e. the promoter is not provided at its native genomic position. .. In certain embodiments, the promoter is genomically integrated into a non-natural genomic position.

In certain embodiments, the nucleic acid encoding the guide RNA is operably linked intracellularly to a regulatory element containing the promoter of the gene of interest. In certain embodiments, the nucleic acid encoding Cas is operably linked intracellularly to a regulatory element containing the promoter of the gene of interest. In certain embodiments, the nucleic acid encoding the guide RNA is operably linked intracellularly to a regulatory element containing the promoter of the gene of interest, and the nucleic acid encoding Cas contains the promoter of the gene of interest. It is operably connected to the regulatory element within the cell. In this latter case, the promoters driving the expression of guide RNA and Cas may be the same or different. In certain embodiments, the nucleic acid encoding the guide RNA and / or Cas is genomically integrated. In certain embodiments, the nucleic acid encoding the guide RNA and / or Cas is extrachromosomal or episomal. The nucleic acid encoding the guide RNA and the nucleic acid encoding Cas may be on the same or different nucleic acid molecules.

The selected DNA sequence targeted by one or more guide RNAs according to the present invention may be an endogenous DNA sequence or an exogenous DNA sequence. Selected DNA sequences targeted by one or more guide RNAs, such as exogenous DNA sequences, may be genomically integrated or extrachromosomal (eg, plasmids) according to the invention. Or provided to the vector). In certain embodiments, the methods as described herein include the step of introducing a vector or plasmid into cells by means known in the art as described elsewhere in the specification. , The vector or plasmid containing the selected DNA sequence and the method comprises detecting a modification of the selected DNA sequence on the vector. It will be appreciated that the vector or plasmid, or at least the selected DNA sequence contained therein, can be genomically integrated, such as by random integration or by homologous recombination. If the target DNA sequence of choice is an endogenous sequence, it is preferred that the sequence be selected such that the modification does not affect the (normal) functionality of the cell or has minimal effect. Those skilled in the art will be able to easily identify such sequences by routine analysis or experimentation. In either case, it is preferred that the endogenous target DNA sequence of such selection is not present in the coding sequence or ORF of the gene and / or in the regulatory sequence of the gene (promoter, enhancer, silencer, etc.).

As described elsewhere herein, the target DNA sequence of choice is a functional CRISPR complex (ie, a guide RNA complexed with Cas protein, where the guide RNA is 5'to 3'. It contains a guide sequence, a tracr mate sequence and a tracr sequence in an orientation, and the tracr sequence may or may not be on the same nucleic acid molecule as the guide sequence and the tracr mate sequence). As used herein, "modified" corresponds to being essentially mutated, i.e., including a point mutation, deletion, substitution, or insertion of one or more nucleotides. As described elsewhere in this specification, the nucleic acid sequence of the target DNA sequence has changed.

However, as described elsewhere herein, "modified" in certain embodiments may not require point mutations, deletions, substitutions, or insertions of one or more nucleotides. It will also be apparent to respond to changes in the target locus, such as activation or suppression of gene transcription, methylation or demethylation of CpG sites. Further, as described elsewhere herein, references to the CRISPR-Cas enzyme as "altering" or "modifying" one or more target polynucleotide loci include, for example, the enzyme itself. Direct changes or modifications due to catalytic activity of, but also indirect changes or modifications due to catalytic activity associated with the CRISPR-Cas enzyme, such as heterologous functional domains, such as transcriptional activation domains. It will also be clear. In addition, as will be understood, one or more target polynucleotide loci that are "altered" or "modified" by the action of the CRISPR-Cas enzyme are, for example, altered or modified by the CRISPR-Cas enzyme itself. It is intended that in embodiments caused by its own catalytic activity, eg, cleavage of DNA by the nuclease activity of the CRISPR-Cas enzyme, it may be included in or adjacent to a polynucleotide sequence complementary to the guide sequence portion of the guide RNA. Will be done. However, an embodiment in which one or more "altered" or "modified" target loci is located at a position different from the sequence complementary to the guide sequence portion of the guide RNA, eg, a heterologous functional domain associated with the CRISPR-Cas enzyme. Also included are embodiments in which alterations or alterations, such as activation or suppression of gene transcription, occur. Thus, "alteration" or "modification" (or similar terminology) of a target locus means that it is due to the direct or indirect action of the CRISPR-Cas enzyme, and further alters or modifies the "target locus". And the "target sequence" complementary to the guide sequence portion of the guide RNA may or may not be the same.

In certain embodiments, the CRISPR-Cas system is multiplexed, ie, a plurality of different guide RNAs may be provided in the methods according to the invention as described herein. Each guide RNA can target (ie, hybridize with) a different selection of DNA targets. Expression of different guide RNAs can be driven by promoters of different genes of interest. Thus, in a particular embodiment, the method of the invention as described herein is a method of determining the expression of two or more, eg, at least two, genes of interest in a cell. Steps to Provide Cells Containing a CRISPR-Cas System (The CRISPR-Cas System comprises two or more, eg, at least two guide RNAs, that target differently selected DNA sequences and Cass capable of modifying the selected DNA sequence. Containing with proteins; thereby each guide RNA is operably linked in the cell to regulatory elements containing promoters of genes of different interests); and said objectives based on the detection of alterations in the DNA sequences of their respective selections. Includes steps to determine the expression of a gene in. In a particular embodiment, two or more different guide RNAs may be operably linked in the cell to a regulatory element containing a promoter of the same gene of interest. Different guide RNAs may be provided on different nucleic acid molecules or on the same nucleic acid molecule. Each guide RNA can be designed such that only modification of the first-choice target DNA produces or destroys the second-choice target DNA.

In certain embodiments, one or more of the components of the CRISPR-Cas system are conditioned (eg, tissue or cell type specific) and / or inducible (eg, chemical inducible) in cells. obtain. Inducible and conditional expression systems are described elsewhere herein. In a detailed embodiment, one or more of the guide RNAs can be conditionally and / or inducibly expressed in cells. In a particularly preferred embodiment, Cas can be conditioned and / or inducibly expressed in cells.

As used herein, the term "targeting" of a selected DNA sequence means that the guide RNA is hybridizable with the selected DNA sequence. As used herein, "hybridization" or "hybridization" is a reaction in which one or more polynucleotides react to form a stable complex by hydrogen bonding between the bases of a nucleotide residue. Point to. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex may include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction can constitute a step in a larger process, such as initiation of PGR or enzymatic cleavage of the polynucleotide. A sequence that is hybridizable with a given sequence is referred to as the "complement" of that given sequence.

As used herein, "expressing" or "expressing" is followed by the process by which a polynucleotide is transcribed from a DNA template (such as to mRNA or other RNA transcript) and / or the transcribed mRNA. Refers to the process of translation into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides are collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells (seil). As used herein, "expression" of a gene or nucleic acid includes not only expression of a cellular gene, but also transcription and translation of one or more nucleic acids in the cloning system and any other context. To.

The terms "polypeptide", "peptide" and "protein" are used herein synonymously to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be fragmented by non-amino acids. These terms also include modified amino acid polymers such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation such as conjugation with a labeling component. As used herein, the term "amino acid" includes both glycine and D or I, optical isomers, and amino acid analogs and peptide mimetics, including natural and / or unnatural or synthetic amino acids. included.

The terms "subject", "individual", and "patient" are used herein synonymously to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murids, primates, humans, agricultural animals, athletic animals, and pets. Also included are tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.

In certain embodiments, the methods and cells according to the invention as described herein can be used in methods of screening and / or diagnosing therapeutic agents. The effect of the temporal expression profile of the candidate therapeutic agent is different, which can be read by the method as described herein.

The terms "therapeutic agent", "therapeutic agent" or "therapeutic agent" are used synonymously to refer to a molecule or compound that imparts some beneficial effect when administered to a subject. Beneficial effects include feasibility of diagnostic judgment; improvement of disease, symptom, disorder, or pathological condition; reduction or prevention of the onset of disease, symptom, disorder or condition; and disease, symptom, disorder or disease. Includes general counter-conditions.

As used herein, "treat" or "treat", or "alleviate" or "improve" are used synonymously. These terms refer to methods for achieving beneficial or desired outcomes, including but not limited to therapeutic and / or prophylactic benefits. Therapeutic benefit means any therapeutically relevant improvement or effect on one or more diseases, conditions, or conditions under treatment. For prophylactic benefits, the composition is a subject at risk of developing a particular disease, condition, or condition, or the physiological condition of the disease, even if the disease, condition, or condition may not yet be present. Can be administered to a subject complaining of one or more of.

As used herein, the terms "chimera RNA," "chimera guide RNA," "guide RNA," "single guide RNA," and "synthetic guide RNA" refer to guide sequences, tracr sequences, and tracr mate sequences. Refers to the containing polynucleotide sequence. The term "guide sequence" refers to an approximately 20 bp sequence within a guide RNA that identifies a target site and can be used synonymously with the term "guide" or "spacer". The term "tracr mate sequence" can also be used synonymously with the term "direct repeat". The guide sequence, tracr, and tracr mate sequence may be provided on a single nucleic acid molecule. Alternatively, the guide and tracr mate sequences may be provided on a single nucleic acid molecule, while tracr is provided on a separate nucleic acid molecule.

Generally, the CRISPR-Cas or CRISPR system is as used in the aforementioned literature, such as WO 2014/093622 (PCT / US2013 / 074667), and collectively, sequences encoding Cas inheritance, tracr (trans-activated CRISPR) sequences (eg, tracrRNA or active partial tracrRNA), tracrmate sequences (including "direct repeats" in the context of endogenous CRISPR systems and partial direct repeats processed by tracrRNA), guide sequences. (Also referred to as a "spacer" in the context of the endogenous CRISPR system), or "RNA" as the term is used herein (eg, a Cas-guided RNA such as Cas9, such as a CRISPR RNA and trans. Involved or active in the expression of CRISPR-related (“Cas”) genes, including activated (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from the CRISPR locus Refers to the transcript and other elements that lead to. In general, the CRISPR system is characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence (also referred to as the protospacer in the context of the endogenous CRISPR system). In the context of the formation of a CRISPR complex, "target sequence" refers to a sequence in which the guide sequence is designed to be complementary to it, where hybridization between the target sequence and the guide sequence is CRISPR. Promotes the formation of complexes. The target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, direct repeats may be identified in silico by searching for repeating motifs that meet some or all of the following criteria: 1. It is present in the genomic sequence of a 2 Kb window adjacent to the type II CRISPR locus; spans 2.20-50 bp; and is only 3.20-50 bp apart. In some embodiments, two of these criteria, such as 1 and 2, 2 and 3, or 1 and 3, may be used. In some embodiments, all three criteria may be used.

In embodiments of the present invention, the term guide sequences and guide RNAs, ie RNAs capable of guiding Cas to target genomic loci, are described in WO 2014/093622 (PCT / US2013 / 074667) and the like. It is used synonymously as described in the above-mentioned cited reference. In general, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and induce sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is approximately 50%, 60%, 75%, 80% when optimally aligned using a suitable alignment algorithm. , 85%, 90%, 95%, 97.5%, 99% or more, or higher. Optimal alignment can be determined using any algorithm suitable for sequence alignment, examples of which are non-limiting examples: Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burroughs-Wheeler transformation based algorithm. (For example, Burroughs Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (available at Novocraft Technologies; www.novocraft.com), ELAND (Illumina, San Digigo, CA), SOAP. (Available) and Maq (available at maq.Sourceforge.net). In some embodiments, the guide sequences are approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer, or longer. In some embodiments, the guide sequence is about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length or less, or shorter. Preferably the guide sequence is 1030 nucleotides in length. The ability of the guide sequence to induce sequence-specific binding of the CRISPR complex to the target sequence can be assessed by any suitable assay. For example, transfection of a vector encoding a component of a CRISPR sequence into a host cell that has a corresponding target sequence, including a guide sequence to be tested, with sufficient components of the CRISPR system to form the CRISPR complex. It may be provided, for example by transfection, and can subsequently be assessed for preferential cleavage within the target sequence, such as by the Surveyor assay as described herein. Similarly, cleavage of the target polynucleotide sequence provides the target sequence, the components of the CRISPR complex including the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and guides them to be tested. It can be determined in vitro by comparing the binding or cleavage rate in the target sequence between the reactions of the sequence and the control guide sequence. Other assays are possible and will be recalled to those of skill in the art.

A guide sequence, an RNA capable of guiding Cas to a genomic target locus, can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell. Illustrative target sequences include those that are unique in the target genome. For example, for S. pyogenes Cas9, as a unique target sequence in the genome, the Cas9 target site of the formula MMMMMMMMNNNNNNNNNNNNNXGG can be mentioned, where NNNNNNNNNNNNNNXGG (N is A, G, T, or C). ; And X can be anything) appear once in the genome. Unique target sequences in the genome can include the Streptococcus pyogenes (S. pyogenes) Cas9 target site of the formula MMMMMMMMMNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N is A, G, T, or C; and what is X). May be present) appears once in the genome. For Streptococcus thermophilus CRISPR1 Cas9, as a unique target sequence in the genome, the Cas9 target site of the formula MMMMMMMMNNNNNNNNNNNXGAAW can be mentioned, where NNNNNNNNNNNNNXXAGAAW (N is A, G, T). Can be anything; and W is A or T) appears once in the genome. A unique target sequence in the genome can include the Streptococcus thermophilus CRISPR1 Cas9 target site of the formula MMMMMMMMMNNNNNNNNNNXGAAW, where NNNNNNNNNNNNXGAAW (N is A, G, T, or C; May; and W is A or T) appear once in the genome. For S. pyogenes Cas9, as a unique target sequence in the genome, the Cas9 target site of the formula MMMMMMMMNNNNNNNNNNNNNNNXGGXG can be mentioned, where NNNNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) appears once in the genome. Unique target sequences in the genome can include the Streptococcus pyogenes (S. pyogenes) Cas9 target site of the formula MMMMMMMMMNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXXG (N is A, G, T, or C; and X is what. May be present) appears once in the genome. In each of these sequences, the "M" may be A, G, T, or C and may not be considered in identifying the sequence as unique. In some embodiments, the guide sequence is selected so that the degree of secondary structure within the guide sequence is reduced. In some embodiments, about 75%, 50%, 40%, 30%, 25%, 20%, 15 of the nucleotides in the guide sequence are involved in self-complementary base pairing when optimally folded. %, 10%, 5%, 1% or less, or less. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of such an algorithm is mFold as described by Zuker and Steigler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is an online web server RNAfold that uses a centroid structure prediction algorithm developed at the Institute for Theoretical Chemistry at the University of Vienna (eg, AR Grubber et al.). , 2008, Cell 106 (1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62).

In general, the tracr mate sequence includes (1) excision of a guide sequence adjacent to the intracellular tracr mate sequence containing the corresponding tracr sequence; and (2) formation of a CRISPR complex in the target sequence (where CRISPR complex). Includes any sequence that has sufficient complementarity with the tracr sequence to promote one or more of the tracr mate sequences hybridized to the tracr sequence). In general, the degree of complementarity relates to the optimal alignment of the tracr mate sequence and the tracr sequence along the shorter of these two sequences. Optimal alignment can be determined by any suitable alignment algorithm, and secondary structure can be considered, such as self-complementarity within either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity along the shorter of these two lengths between the tracr sequence and the tracr mate sequence when optimally aligned is approximately 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more, or higher. In some embodiments, the tracr sequence is about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,40, It is at least 50 nucleotides in length or longer. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript, and hybridization between the two results in a transcript with secondary structure, such as a hairpin. In certain embodiments of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In a preferred embodiment, the transcript has 2, 3, 4 or 5 hairpins. In a further embodiment of the invention, the transcript has at most 5 hairpins. In the hairpin structure, a portion of the sequence on the 5'side of the last "N" and upstream of the loop corresponds to the tracr mate sequence, and a portion of the sequence on the 3'side of the loop corresponds to the tracr sequence. Further non-limiting examples of single polynucleotides comprising guide sequences, tracr mate sequences, and tracr sequences are as follows (listed in order from 5'to 3'), where "N" is the guide sequence. The first lowercase block represents the tracr mate sequence, the second lowercase block represents the tracr sequence, and the last poly T sequence represents the transcription terminator:
In some embodiments, sequences (1)-(3) are used in combination with Cas9 of Streptococcus thermophilus CRISPR1. In some embodiments, sequences (4)-(6) are used in combination with Cas9 of Streptococcus pyogenes (S. pyogenes). In some embodiments, the tracr sequence is a transcript separate from the transcript containing the tracr mate sequence.

In some embodiments, candidate tracrRNAs can then be predicted by sequences that meet some or all of the following criteria: 1. Sequence homology for direct repeat (motif search with a maximum of 18 bp mismatch in Geneius); 2. Presence of expected Rho-independent transcription terminators in the transcription direction; and 3. Stable hairpin secondary structure between tracrRNA and direct repeat. In some embodiments, two of these criteria, such as 1 and 2, 2 and 3, or 1 and 3, may be used. In some embodiments, all three criteria may be used.

In some embodiments, the chimeric synthesis guide RNA (sgRNA) design can incorporate at least 12 bp of double-stranded structure between the direct repeat and the tracrRNA.

RNA for guide Cas, eg Cas9, can include CRISPR RNA and transactivated (tracr) RNA. The tracr mate sequence and the tracr sequence can be combined to form a transactivating (tracer) sequence. The tracr mate and tracr sequences can be optionally designed to form a single guide RNA (sgRNA). In fact, it is advantageous that the RNA for guide Cas can include a chimeric single guide RNA (sgRNA). The optimally aligned tracr sequence and tracr mate sequence are approximately 25%, 30%, 40%, 50%, 60%, 70%, 80% along the shorter of these two lengths. , 90%, 95%, 97.5%, 99% or more, or higher. Is the tracr sequence greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 nucleotides in length? , Or longer.

In the classical CRISPR-Cas system, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97. .5%, 99%, or 100% or more; guide or RNA or sgRNA is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer, or longer; or the guide or RNA or sgRNA It may be less than or shorter than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length; and advantageously the tracrRNA is 30 or 50 nucleotides in length. However, one aspect of the invention is to reduce off-target interactions, eg, reduce guide interactions with less complementary target sequences. In fact, in these examples, between target and off-target sequences having 80% to greater than about 95% complementarity, eg, 83% to 84% or 88 to 89% or 94 to 95% complementarity. Mutations that result in a CRISPR-Cas system that can be distinguished (eg, distinguishing targets with 18 nucleotides from off-targets with 18 nucleotides with 1, 2 or 3 mismatches) are involved in the invention. Shown. Therefore, in the context of the present invention, the degree of complementarity between the guide sequence and its corresponding target sequence is 94.5% or 95% or 95.5% or 96% or 96.5% or 97%. Or 97.5% or 98% or 98.5% or 99% or 99.5% or more than 99.9%, or 100%. Off-targets are 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% between the sequence and the guide. Or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or Complementarity of less than 83% or 82% or 81% or 80%, advantageously off-target is 100% or 99.9% or 99.5% or 99% or 99 between its sequence and guide. % Or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity.

In a particularly preferred embodiment of the invention, the guide RNA (having the ability to guide Cas to the target locus) is (1) a guide sequence capable of hybridizing to the genomic target locus of eukaryotic cells; (2) tracr. Sequences; and (3) tracr mate sequences may be included. All (1) to (3) may be in a single RNA (arranged in the direction from 5'to 3'), i.e. in an sgRNA, or the tracr RNA is an RNA encoding a guide and tracr sequence. It may be an RNA different from. The tracr hybridizes to the tracr mate sequence and directs the CRISPR / Cas complex to the target sequence.

The methods according to the invention as described herein include eukaryotic cells as discussed herein (in vitro,) comprising delivering the vector as discussed herein to the cell. That is, it involves the step of inducing one or more mutations in (isolated eukaryotic cells). This one or more mutations include the introduction, deletion, or substitution of one or more nucleotides in each target sequence of one or more cells via one or more guide RNAs or sgRNAs. obtain. This mutation may include the introduction, deletion, or substitution of 1-75 nucleotides in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. This mutation includes 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides. This mutation includes 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides. This mutation includes 10,11,12,13,14,15,16,17,18,19, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides. This mutation includes 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, in each target sequence of one or more of the cells via one or more guide RNAs or sgRNAs. It may include the introduction, deletion, or substitution of 30, 35, 40, 45, 50, or 75 nucleotides. The mutation involves the introduction of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides in each target sequence of the cell via one or more guide RNAs or sgRNAs. , Deletion, or substitution can be included.

It may be important to control the concentration of Cas mRNA and guide RNA delivered to minimize toxicity and off-target effects. Optimal concentrations of Cas mRNA and guide RNA should be determined by testing various concentrations in cellular or non-human eukaryotic animal models and analyzing the degree of alteration at potential off-target genomic loci using deep sequencing. Can be done. Alternatively, to minimize the level of toxicity and off-target effects, Cas nickase mRNA (eg, S. pyogenes Cas9 with a D10A mutation) is used as a pair of guide RNAs that target the site of interest. Can be delivered with. Guide sequences and strategies for minimizing toxicity and off-target effects may be as described in WO 2014/093622 (PCT / US2013 / 074667); or herein. It may be mutated as described in.

In some embodiments, the CRISPR system is advantageously derived from the type II CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism, including the endogenous CRISPR system, eg, Streptococcus pyogenes. In a preferred embodiment of the invention, the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes the cleavage of DNA. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1. , Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx1 , Csx15, Csf1, Csf2, Csf3, Csf4, their homologues, or modified forms thereof. The preferred Cas enzyme can be identified as Cas9 because Cas9 can refer to a general enzyme class that shares homology with the largest nuclease containing multiple nuclease domains of the type II CRISPR system. Most preferably, the Cas9 enzyme is derived from or derived from SpCas9 or SaCas9. It will be understood that SpCas9 or SaCas9 is derived from or derived from S. pyogenes or S. aureus Cas9. Induced is based on the wild-type enzyme in the sense that the derived enzyme generally has a high degree of sequence homology with it, but it is mutated in some way as described herein. It means that it is (modified). It will be understood that the terms Cas and CRISPR enzymes are generally used interchangeably herein, unless otherwise specified. The Cas enzyme may be, for example, any naturally occurring bacterial Cas9 and any chimera, mutant, homolog or ortholog. Many of the residue numbering used herein is based on the Cas9 enzyme at the type II CRISPR locus in Streptococcus pyogenes (or annotated with SpCas9 or spCas9). However, the present invention includes more Cas9s from other microbial species, such as SpCas9 orthologs, or S. pyogenes plus microbially derived Cas9s, such as Staphylococcus aureus (S. It will be understood that it includes SaCas9 derived from aureus), St1Cas9 derived from S. thermophilus, and the like. One of ordinary skill in the art will be able to determine the appropriate corresponding residue in Cas9 enzymes other than SpCas9 by comparing the relevant amino acid sequences. Therefore, when a particular amino acid substitution is referenced using SpCas9 numbering, the disclosure includes corresponding modifications in other Cas9 enzymes, unless it is clear in the context that it is not intended to refer to another Cas9 enzyme. Is intended to be.

In some embodiments, unmodified CAS, such as Cas9, has DNA-cleaving activity. In some embodiments, CAS induces cleavage of one or both strands within the target sequence and / or within the complement of the target sequence. In some embodiments, the CAS is from the first or last nucleotide of the target sequence, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8. Induces cleavage of one or both strands within one, about 9, about 10, about 15, about 20, about 25, about 50, about 100, about 200, about 500, or more base pairs. In some embodiments, the vector is a CAS mutated to the corresponding wild-type enzyme such that the mutated CAS loses the ability to cleave one or both strands of the target polynucleotide containing the target sequence. Code. For example, the replacement of aspartic acid with alanine (D10A) in the RuvCI catalytic domain of Cas9 from Streptococcus pyogenes (S. pyogenes) causes nickase (single-strand breaks) from a nuclease that cleaves both chains of Cas9. Convert to. Other examples of mutations that make Cas9 nickase include, but are not limited to, H840A, N854A, and N863A. As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III, or HNH domains) are mutated to create mutant Cas9 that has substantially lost all DNA cleavage activity. Can be made to. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to create a Cas9 enzyme that has substantially lost all DNA cleavage activity. In some embodiments, the CAS has a mutational enzyme DNA cleavage activity of about 25% or less, about 10% or less, about 5% or less, about 1% or less of the non-mutant enzyme DNA cleavage activity. When it is about 0.1% or less, about 0.01% or less, or less, it is considered that all DNA cleavage activity is substantially lost; one example is that the mutant DNA cleavage activity is , Zero, or when it is negligible compared to the non-mutated form. Thus, Cas may contain one or more mutations and can be used as a common DNA binding protein with or without fusion to a functional domain. The mutation may be an artificially introduced mutation or a gain-of-function or loss-of-function mutation. Mutations can include, but are not limited to, mutations in one of the catalytic domains in the RuvC and HNH catalytic domains (eg, D10 and H840); or the CRISPR enzymes are D10A, E762A, H840A, N854A. Can include one or more mutations selected from the group consisting of, N863A or D986A and / or one or more mutations in the RuvC1 or HNH domain of Cas, or as otherwise discussed herein. Has a mutation. In one aspect of the invention, the Cas enzyme may be fused to a protein such as TAG and / or an inducible / controllable domain such as a chemically inducible / controllable domain. The Cas in the present invention may be a chimeric Cas protein; for example, Cas whose function has been enhanced by being chimeric. The chimeric Cas protein can be a novel Cas containing fragments from two or more naturally occurring Cass. Such a chimeric Cas protein may comprise a fusion of one or more N-terminal fragments of one Cas9 homolog and one or more C-terminal fragments of another Cas homolog. Cas can be delivered to cells in the form of mRNA. Expression of Cas may be under the control of an inducible promoter. If the enzyme is not SpCas9, mutations can occur at some or all residues corresponding to positions 10, 762, 840, 854, 863, and / or 986 of SpCas9 ( For example, it can be confirmed by a standard sequence comparison tool). In particular, some or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A, and / or D986A; and conservative substitutions of any of the substituted amino acids are also contemplated. The same (or conservative) substitution of these mutations at the corresponding positions in other Cas9s is also preferred. D10 and H840 in SpCas9 are particularly preferred. However, for other Cas9s, residues corresponding to SpCas9 D10 and H840 are also preferred. It is expressly an object of the present invention to avoid reading into known mutations. That is, Cas9 is nickase or has little or no nuclease activity when compared to, for example, non-mutated Cas9, eg 5% or less than 5%, eg 4%, 3%, 2% or 1%. Mutations known in the art to be less than "dead" are intended to be within the Cas9 mutations that reduce or eliminate the interaction between the guide and the off-target nucleic acid molecule. However, Applicants reserve the right to use the proviso to exclude mutations that result in such known "nickase" or "dead" Cas9. In fact, the phrase "then the enzyme in the CRISPR complex is less capable of modifying one or more off-target loci when compared to the unmodified enzyme, and / or thereby the enzyme in the CRISPR complex is the unmodified enzyme. "Increased ability to modify one or more target loci when compared" (or similar expression) is not intended to be read as a mutation that only results in nickase or dead Cas9 or a known Cas9 mutation. However, this means that "then the enzyme in the CRISPR complex is less capable of modifying one or more off-target loci when compared to the unmodified enzyme, and / or thereby the enzyme in the CRISPR complex is unmodified. One or more modifications or mutations of the present invention, such as "increased ability to modify one or more target loci when compared to an enzyme" (or similar expression), with nickase or dead. I'm not saying that it can't be combined with mutations that result in an enzyme. Such dead enzymes can be enhanced nucleic acid molecular binding agents. And such nickases can be enhanced nickases. For example, one or more neutral amino acids and / or nucleic acids in and / or in the vicinity thereof (eg, other charged residues at other positions in Cas9 very close to DNA, cDNA, RNA, sgRNA). Changing to one or more positively charged amino acids "thereby reducing the ability of the enzyme in the CRISPR complex to modify one or more off-target loci when compared to unmodified enzymes, and / or thereby reducing the ability of the enzyme to modify one or more off-target loci. Enzymes in the complex have an increased ability to modify one or more target loci when compared to unmodified enzymes, which can result in, for example, further cleavage, which is enhanced on-target cleavage. And because it can be both off-target cleavage (supercutting Cas9), by using it with what is known in the art as a true-guide or true-sgRNA (eg, Fu et al., "Transited guide RNA". CRISPR-Cas nuclease specificity improvement (improving CRISPR-Cas sensifectivity using CRISPR-Cas nuclease specific RNAs) ”, Nature Biotechnology 32,279-284 (2014) doive10 January 2014 Online Publication 26 January 2014 Directed online 29 January 2014) Enhances on-target activity without increased off-target cleavage, or creates super-cutting nickase, or dead Cas9 for super-binding agent Combine with mutation.

SpCas9 orthologs can be used in the practice of the present invention. The Cas enzyme can be the identified Cas9 because it can refer to a common class of enzyme that shares homology with the largest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from or is derived from spCas9 (Streptococcus pyogenes Cas9) or saCas9 (Staphylococcus aureus Cas9). "StCas9" refers to wild-type Cas9 from Streptococcus thermophilus, and this protein sequence is present in the SwissProt database as accession number G3ECR1. Similarly, S. pyogenes Cas9 or spCas9 , Is stored in the SwissProt database as accession number Q99ZW2. Origin is largely based on wild-type enzymes in that the derived enzymes have a high degree of sequence homology with wild-type enzymes. However, it means that it has been mutated (modified) in any way described herein. The terms Cas and CRISPR enzymes are generally used herein unless explicitly stated. It should be understood that they are used interchangeably. As mentioned above, many of the residue numbering used herein is the Cas9 enzyme from the type II CRISPR locus in Streptococcus thermogenes. However, it should be understood that the present invention includes more Cas9 from other species of microorganisms, such as SpCas9, SaCa9, and St1Cas9. Cas9 from Streptococcus pyogenes or any of them. The enzymatic action of Cas9, which is closely related to, results in double-strand breaks in the sequence of the target site that hybridizes to the 20 nucleotides of the guide sequence, which is the protospacer flanking motif (PAM) following the 20 nucleotides. ) Sequence (eg, NGG / NRG or PAM which can be determined as described herein). CRISPR activity by Cas9 for site-specific DNA recognition and cleavage is a guide sequence, The CRISPR activity by Cas9 for site-specific DNA recognition and cleavage is determined by the tracr sequence that partially hybridizes to this guide sequence, and the tracr that partially hybridizes to the guide sequence. Defined by sequence and PAM sequence. Although not desired to be constrained by theory, we believe that the target sequence must be accompanied by a PAM (protospacer flanking motif), a short sequence recognized by the CRISPR complex. The exact sequence and length requirements for PAM will vary depending on the Cas used, but PA M is typically a protospacer (ie, a sequence of 2-5 base pairs adjacent to the target sequence. In some embodiments, the method comprises attaching the CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex is: , Cas containing a complex with a guide sequence that hybridizes to the target sequence within the target polynucleotide, the guide sequence is linked to the tracr mate sequence, and then the tracr mate sequence hybridizes to the tracr sequence. A further aspect of the CRISPR system is Karginov and Hannon, "CRISPR system: small RNA-guided defense in bacteria and archaea (The CRISPR system: small RNA-guided defense in bacteria and archaea)", Mole Cell. 37 (1): 7. The type II CRISPR locus from Streptococcus pyogenes SF370 is a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA, and short length non-repeating sequences. Includes a characteristic array of repetitive sequences (direct repeats) with (spacers, each about 30 bp) inserted in between. In this system, target DNA double-strand breaks (DSBs) are performed in four consecutive steps. First, two non-coding RNAs, a pre-crRNA array, and a tracrRNA are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to a direct repeat of the pre-crRNA, and then the hybridized pre-crRNA is processed into a mature crRNA containing the individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs Cas to a DNA target consisting of the protospacer and the corresponding PAM by heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas mediates the cleavage of target DNA upstream of PAM to give rise to DSB in the protospacer. A pre-crRNA array consisting of a single spacer flanking two direct repeats (DRs) is also included in the term "tracrmate sequence". In certain embodiments, Cas can be constitutively present, or inductively present, conditionally present, administered, or delivered. Cas optimization can be used to develop new features that can enhance functionality or produce chimeric Cas proteins. And Cas can be used as a common DNA binding protein.

Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex, including a guide sequence that hybridizes to the target sequence to form a complex with one or more Cas proteins, in or without the formation of the target sequence. Neighboring range (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence (Inside) one or both chains are cut. Without wishing to be bound by theory, the tracr sequence contains all or part of the wild-type tracr sequence or all or part of it (eg, about 20, about 26, about 32 of the wild-type tracr sequence, About 45, about 48, about 54, about 63, about 67, about 85, or more, or more than about 20, more than about 26, more than about 32, more than about 45, more than about 48, Can consist of more than about 54, more than about 63, more than about 67, more than about 85, or more nucleotides) and is functional to the guide sequence, eg, along at least part of the tracr sequence. Part of the CRISPR complex may also be formed by hybridization to all or part of the tracr mate sequence linked to.

In the present disclosure, the term "Cas" may mean "Cas9" or the CRISPR enzyme. In the context of the present invention, the Cas9 or Cas or CRISPR enzyme is mutated or modified so that the enzyme in the CRISPR complex has the ability to modify one or more off-target loci when compared to the unmodified enzyme. "Decrease" (or similar expression); and when reading this specification, terms such as "Cas9" or "Cas" or "CRISPR enzyme" are mutated or modified Cas9 or Cas or CRISPR in the present invention. It is intended to include an enzyme, i.e., "it reduces the ability of an enzyme in the CRISPR complex to modify one or more off-target loci when compared to an unmodified enzyme" (or similar expression).

Codon Optimization and Codon Use for Expression of Cas Protein The nucleic acid molecule encoding Cas is advantageously codon-optimized Cas. An example of a codon-optimized sequence is, in this case, a eukaryotic, eg, a sequence whose expression in humans is optimized (ie, expression in humans is optimized), or described herein. Is an optimized sequence for expression in another eukaryotic, animal, or mammal; eg, SaCas9 human codon optimal in WO 2014/093622 (International Application PCT / US2013 / 074667). Please refer to the conversion sequence. This is preferred, but it should be understood that other examples are possible and that codon optimizations of other host species other than humans, or codon optimizations of specific organisms, are also known. In some embodiments, the enzyme coding sequence encoding Cas is a codon whose expression is optimized in a particular cell, eg, a eukaryotic cell. Eukaryotes are specific organisms, such as mammals, including but not limited to humans, or non-human eukaryotic animals, animals, or mammals described herein, such as mice, rats, rabbits. , Dogs, livestock, or non-human mammals or primates, or derived from them. In some embodiments, the process of altering the genetic identity of a human germline and / or an animal that has no substantial medical advantage to the human or animal and is likely to afflict the human or animal. The process of altering genetic identity, as well as the animals resulting from such a process, can be excluded. In general, codon optimization involves at least one codon of the natural sequence (eg, about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 25). 50 or more, or more than about 1, more than about 2, more than about 3, more than about 4, more than about 4, more than about 5, more than about 10, more than about 15, about More than 20, about 25, more than about 50, more codons) are replaced with the more frequently or most frequently used codons used in the genes of the host cell, while Refers to the process of modifying a nucleic acid sequence to promote expression in a host cell of interest by maintaining the natural amino acid sequence. Various species exhibit a particular bias towards a codon of a particular amino acid. Codon bias (difference in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which, among other things, is the characteristics of the codons being translated and the particular transfer RNA (tRNA) molecule. It is considered to depend on availability. The predominance of selected tRNAs in cells is generally a reflection of the most frequently used codons in peptide synthesis. Thus, genes can be tuned for optimal gene expression in a given organism based on codon optimization. The codon usage frequency table is, for example, www. kazusa. readily available in the "Codon Usage Database" available at orjp / codon /, these tables can be adapted in a number of ways. Nakamura, Y. et al. , Et al. "Codon usage databased from the international DNA sequence database: status for the year 2000" Nucl. Acids Res. See 28: 292 (2000). Computer algorithms for codon-optimizing specific sequences for expression in specific host cells are also available, and for example, Gene Forge (Aptagen; Jacobus, PA) are also available. In some embodiments, one or more codons (eg, one, two, three, four, five, ten, 15, 20, 25, 50, or more) of the sequence encoding Cas. Many or all codons) correspond to the codons most frequently used for a particular amino acid.

In certain embodiments, the methods as described herein encode one or more guide RNAs operably linked intracellularly to a regulatory element containing the promoter of one or more genes of interest. It may include the step of providing Cas transgenic cells into which one or more nucleic acids are provided or introduced. As used herein, the term "Cas transgenic cell" refers to a cell, such as a eukaryotic cell, in which the Cas gene is genomically integrated. The nature, type, or origin of the cell is not particularly limited according to the present invention. The method for introducing the Cas trans gene into a cell may also be various and may be any method known in the art. In certain embodiments, Cas transgenic cells are obtained by introducing the Cas transgenic gene into isolated cells. In certain other embodiments, Cas transgenic cells are obtained by isolating the cells from a Cas transgenic organism. As an example and without limitation, Cas transgenic cells as referred to herein may be derived from Cas transgenic eukaryotes, such as Cas knock-in eukaryotes. International Publication No. 2014/03622 Pamphlet (PCT / US13 / 74667) (incorporated herein by reference) is referenced. Sangamo BioSciences, Inc. on targeting the Rosa locus. The methods of U.S. Patent Application Publication No. 20120017290 and No. 20110265198 assigned to the United States patent application can be modified to utilize the CRISPR Cas system of the present invention. The method of U.S. Patent Application Publication No. 201302336946, assigned to Celtecis for targeting the Rosa locus, can also be modified to utilize the CRISPR Cas system of the invention. As a further example, Platt et., Which describes a Cas9 knock-in mouse. al. (Cell; 159 (2): 440-455 (2014)) (incorporated herein by reference). The Cas trans gene may further comprise a Lox-Stop-poly A-Lox (LSL) cassette, whereby Cas expression can be induced by Cre recombinase. Alternatively, Cas transgenic cells may be obtained by introducing the Cas trans gene into isolated cells. Transgene delivery systems are well known in the art. As an example, the Cas trans gene is similarly described elsewhere herein, for example in eukaryotic cells with vectors (eg, AAV, adenovirus, lentivirus) and / or particle and / or nanoparticle delivery. Can be delivered using.

Those skilled in the art will appreciate that cells such as Cas transgenic cells as referred to herein contain additional genomic alterations in addition to having the integrated Cas gene, or, for example, and without limitation, Platt et al. .. (2014), Chen et al. , (2014) or Kumar et al. .. As described in (2009), mutations caused by the sequence-specific action of Cas when complexed with RNA capable of guiding Cas to a target locus, such as one or more carcinogenic mutations. You will understand that it can be included.

Cas protein with one or more NLS In some embodiments, the Cas sequence is one or more nuclear localization sequences (NLS), eg, about one, about two, about three, about four. , About 5, about 6, about 7, about 8, about 9, about 10, or more, or more than about 1, more than about 2, more than about 3, more than about 3 Fuse into more than four, more than about five, more than about six, more than about seven, more than about eight, more than about nine, more than about ten, or more than NLS. In some embodiments, Cas is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, near the amino terminus or its vicinity. One, about ten or more, or more than about one, more than about two, more than about three, more than about four, more than about five, more than about six, about seven More than one, more than about eight, more than about nine, more than about ten, or more than NLS, about one, about two, about three, about four near the carboxy terminus, About 5, about 6, about 7, about 8, about 9, about 10, or more, or more than about 1, more than about 2, more than about 3, more than about 4, about 4 More than 1, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 9, more than about 10, or more NLS, or a combination thereof (For example, 0 or at least one NLS at the amino terminus, and 0 or one or more NLS at the carboxy terminus). If more than one NLS is present, each is independent of the other so that a single NLS can be present in more than one copy, and / or one or more present in one or more copies. It can be selected in combination with other NLS. In a preferred embodiment of the invention, the CRISPR enzyme comprises up to 6 NLS. In some embodiments, the NLS has the closest amino acid of this NLS from the N-terminus or C-terminus along the polypeptide chain to about 1, 2, 3, 4, 5, 5, 10, 15, If it is in 20, 25, 30, 40, 50, or more amino acids, it is considered to be near the N-terminus or C-terminus. A non-limiting example of NLS is the NLS of the SV40 viral large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: X); the NLS from the nuclearoplasmin (eg, the two-part nuclearoplasmin having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: X) (eg). bipartite) NLS); amino acid sequence PAAKRVKLD (SEQ ID NO: X) or RQRRNELKRSP (SEQ ID NO: X) c-myc NLS having; hRNPA1 M9 NLS having a sequence EnukyuesuesuenuefuJipiemukeijijienuFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: X); sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of IBB domains from importin alpha ( SEQ ID NO: X); Myoma T protein sequences VSRKRPRP (SEQ ID NO: X) and PPKKARED (SEQ ID NO: X); Human p53 sequence POPKKKPL (SEQ ID NO: X); Mouse c-abl IV sequence SALIKKKKKKMAP (SEQ ID NO: X); Influenza Virus NS1 sequence DRLRR (SEQ ID NO: X) and PKQKKRK (SEQ ID NO: X); Hepatitis virus δ antigen sequence RKLKKKIKKL (SEQ ID NO: X); Mouse Mx1 protein sequence REKKKFLKRR (SEQ ID NO: X); Human poly (ADP-ribose) polymerase Sequence KRKGDEVDGVDEVAKKKKSK (SEQ ID NO: X); and NLS sequence derived from the steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK (SEQ ID NO: X). In general, one or more NLSs are strong enough to accumulate a detectable amount of Cas in the nucleus of a eukaryotic cell. In general, the intensity of nuclear localization activity can arise from the number of NLS in Cas, the particular NLS used, or a combination of those factors. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to Cas, thereby combining the intracellular location with, for example, a means for detecting the location of the nucleus (eg, a nucleus-specific stain, eg, DAPI). It can be visualized with. Cell nuclei can also be isolated from cells and their contents can then be analyzed by any suitable process for detecting proteins, such as immunohistochemical analysis, Western blot, or enzyme activity assay. Accumulation in the nucleus is affected, for example, by an assay for the effect of CRISPR complex formation (eg, an assay for cleavage or mutation of DNA in a target sequence, or CRISPR complex formation and / or Cas activity. Assayed for gene expression activity) to determine indirectly compared to controls exposed to Cas that has not been exposed to Cas or complex, or has been exposed to Cas lacking one or more NLS. You can also. In some embodiments, NLS is not added or fused to the Cas protein.

Delivery of CRISPR Systems Using this disclosure and knowledge in the art, the CRISPR-Cas systems, in particular the novel CRISPR systems described herein, or their components or nucleic acid molecules thereof (including, for example, HDR templates) or theirs. Nucleic acid molecules encoding or providing components can be delivered by the delivery system described herein in general and in detail.

General Information About Vectors In general, and throughout the specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; free-ended (eg, circular) nucleic acid molecules containing one or more free ends; DNA, Nucleic acid molecules containing RNA, or both; and other types of polynucleotides known in the art. Certain vectors are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where the vector is a virus for packaging into viruses (eg, retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus). The derived DNA or RNA sequence is present. Viral vectors also include polynucleotides carried by the virus for transfection into host cells. A particular vector is capable of self-renewal in the host cell into which it is introduced (eg, a bacterial vector with a bacterial origin of replication and an episomal mammalian vector). Other vectors (eg, non-episome mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. In addition, a particular vector can guide the expression of a gene to which it is operably linked. Vectors for and resulting in expression in eukaryotic cells can be referred to herein as "eukaryotic expression vectors." Such vectors are referred to herein as "expression vectors". Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

The recombinant expression vector can contain a form of nucleic acid of the invention suitable for expression of the nucleic acid in the host cell, that is, one in which the recombinant expression vector is operably linked to the nucleic acid sequence to be expressed. It is meant to include the above regulatory elements, which may be selected based on the host cell used for expression. Within the scope of recombinant expression vectors, "operably linked" allows expression of nucleotide sequences (eg, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell). It is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements.

The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, polyadenylation signals and transcription termination signals such as polyU sequences). .. For such adjustment elements, see, for example, Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that induce constitutive expression of nucleotide sequences in many types of host cells and those that induce expression of nucleotide sequences only in specific host cells (eg, tissue-specific regulatory sequences). Tissue-specific promoters are primarily expressed in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg liver, pancreas), or specific cell types (eg lymphocytes). Can be derived. Regulatory elements may also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, and this expression may or may not be tissue- or cell-type specific. In some embodiments, the vector is one or more pol III promoters (eg, 1, 2, 3, 4, 5, or more pol III promoters) and one or more pol II promoters (eg, 1, 2, 3, 4, 5, or more). One, two, three, four, five or more pol II promoters), one or more pol I promoters (eg, 1, 2, 3, 4, 5, or more pol I promoters), Or includes a combination of these. Examples of the pol III promoter include, but are not limited to, the U6 and H1 promoters. Examples of the pol II promoter include, but are not limited to, the retrovirus Raus sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [eg, with the CMV enhancer). See Boshard et al, Cell, 41: 521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. In addition, the term "adjustment element" includes an enhancer element such as WPRE; CMV enhancer; R-U5'segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8 (1), p. 466-472). , 1988); SV40 enhancer; and intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78 (3), p. 1527-31, 1981). Is also included. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to transform, the desired expression level, and the like. The vector can be introduced into a host cell, whereby the transcript, protein, or peptide encoded by the nucleic acid as described herein is a fusion protein or peptide (eg, clustered regular). Shortly spaced Palindrome repeat (CRISPR) transcripts, proteins, enzymes, mutants thereof, fusion proteins thereof, etc.) can be produced.

Advantageous vectors include lentivirus and adeno-associated virus, and the type of such vector can also be selected for targeting specific types of cells.

In some embodiments, one or more vectors driving the expression of one or more elements of the nucleic acid targeting system are introduced into the host cell, and when the elements of the nucleic acid targeting system are expressed, the nucleic acid at one or more target sites. The formation of the targeting complex is guided. For example, the nucleic acid targeting effector enzyme and the nucleic acid targeting guide RNA may be operably linked to separate regulatory elements on separate vectors. One or more RNAs of the nucleic acid targeting system are transgenic nucleic acid targeting effector protein animals or mammals, eg, animals or mammals that constitutively or inducibly or conditionally express the nucleic acid targeting effector protein; or by nature. Animals or mammals expressing or having cells containing the nucleic acid targeting effector protein are pre-administered to it one or more vectors encoding and expressing the nucleic acid targeting effector protein in vivo. And so on. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined into a single vector, with one or more additional vectors not included in the first vector of nucleic acid targeting systems. Provide any component. Nucleic acid targeting elements combined into a single vector have one element located 5'side ("upstream" of it) or 3'side ("downstream") of the second element. It may be arranged in any suitable orientation, such as being positioned. The coding sequence of one element may be located on the same chain or reverse chain as the coding sequence of the second element, and may be oriented in the same or opposite direction. In some embodiments, a single promoter is integrated within one or more intron sequences (eg, each in a different intron, two or more in at least one intron, or all). Drives the expression of transcripts encoding nucleic acid targeting effector proteins and nucleic acid targeting guide RNAs (in a single intron). In some embodiments, the nucleic acid targeting effector protein and the nucleic acid targeting guide RNA are operably linked to the same promoter and may be expressed from there. For delivery media, vectors, particles, nanoparticles, formulations and their constituents for expressing one or more elements of the nucleic acid targeting system, see WO 2014/093622 (PCT / US2013 / 074667). Etc., as used in the above-mentioned literature. In some embodiments, the vector comprises one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, one or more insertion sites (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or more insertion sites) are one. It is located upstream and / or downstream of one or more sequence elements of the above vector. In some embodiments, the vector comprises an insertion site upstream of the tracr mate sequence and, optionally downstream of the regulatory element operably linked to the tracr mate sequence, after insertion of the guide sequence into the insertion site. And upon expression, the guide sequence leads to sequence-specific binding of the nucleic acid targeting complex to the target sequence in eukaryotic cells. In some embodiments, the vector contains more than one insertion site, allowing the insertion of a guide sequence into each site. In such a configuration, the two or more guide sequences may include two or more copies of a single guide sequence, two or more different guide sequences, or a combination thereof. Using multiple different guide sequences, a single expression construct can be used to target nucleic acid targeting activity to multiple different corresponding target sequences within the cell. For example, a single vector may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more guide sequences, or more. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more such guide sequence-containing vectors are provided, and optionally delivered to cells. May be done. In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence encoding a nucleic acid targeting effector protein. Nucleic acid targeting effector proteins or one or more nucleic acid targeting guide RNAs may be delivered separately; and advantageously, at least one of these is delivered by a particle or nanoparticle complex. Nucleic acid targeting effector protein mRNA may be delivered prior to nucleic acid targeting guide RNA to give time for expression on the nucleic acid targeting effector protein. Nucleic acid targeting effector protein mRNA may be administered 1 to 12 hours (preferably about 2 to 6 hours) prior to administration of the nucleic acid targeting guide RNA. Alternatively, the nucleic acid targeting effector protein mRNA and the nucleic acid targeting guide RNA may be administered together. Advantageously, a second booster dose of guide RNA may be administered 1-12 hours (preferably about 2-6 hours) after the initial administration of the nucleic acid targeting effector protein mRNA + guide RNA. Additional administration of nucleic acid targeting effector protein mRNA and / or guide RNA may be useful in achieving the most efficient levels of genomic modification.

General Information Regarding Vector Delivery In certain embodiments, the present invention is used, for example, to deliver or introduce into cells an RNA capable of guiding Cas and / or Cas to a target locus (ie, a guide RNA). It also relates to vectors that are also for the growth of these components (eg, in prokaryotic cells). As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. This is a replicon, such as a plasmid, phage, or cosmid, on which another DNA segment can be inserted to result in replication of the inserted segment. In general, the vector is replicable when associated with the appropriate control element. In general, and throughout the specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; free-ended (eg, circular) nucleic acid molecules containing one or more free ends; DNA, Nucleic acid molecules containing RNA, or both; and other types of polynucleotides known in the art. Certain vectors are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where the vector is of packaging into a virus (eg, retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus (AAV)). There is a virus-derived DNA or RNA sequence for this. Viral vectors also include polynucleotides carried by the virus for transfection into host cells. A particular vector is capable of self-renewal in the host cell into which it is introduced (eg, a bacterial vector with a bacterial origin of replication and an episomal mammalian vector). Other vectors (eg, non-episome mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. In addition, a particular vector can guide the expression of a gene to which it is operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

The recombinant expression vector can contain a form of nucleic acid of the invention suitable for expression of the nucleic acid in the host cell, that is, one in which the recombinant expression vector is operably linked to the nucleic acid sequence to be expressed. It is meant to include the above regulatory elements, which may be selected based on the host cell used for expression. Within the scope of recombinant expression vectors, "operably linked" allows expression of nucleotide sequences (eg, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell). It is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements. Regarding recombination and cloning methods, U.S. Patent Application No. 10 / 815,730, published as U.S. Patent Application Publication No. 2004-0171156 A1 on September 2, 2004 (this content is described herein). Incorporated by reference as a whole in the book).

One or more vectors may contain one or more regulatory elements, such as one or more promoters. One or more vectors may contain Cas coding sequences and / or a single but at least 3 or 8 or 16 or 32 or 48 or 50 guide RNA (eg sgRNA) coding sequences, eg, 1-2, 1-3, 1-4 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32 It may contain 3 to 48, 3 to 50 RNAs (eg, sgRNA). In a single vector, each RNA (eg, sgRNA) may have a promoter, provided that there are up to about 16 RNAs (eg, sgRNA) advantageously; and RNA with more than 16 single vectors (eg, sgRNA). For example, when providing sgRNA), one or more promoters may drive the expression of two or more RNAs (eg, sgRNA), for example, if there are 32 RNAs (eg, sgRNA), each promoter is 2 Expression of RNA (eg, sgRNA) may be driven, and if there are 48 RNAs (eg, sgRNA), each promoter may drive expression of 3 RNAs (eg, sgRNA). With a simple mathematically well-established cloning protocol and the teachings of the present disclosure, one of ordinary skill in the art will appreciate RNA (eg, sgRNA for a suitable exemplary vector such as AAV, and a suitable promoter such as the U6 promoter, eg U6- The present invention can be readily practiced with respect to sgRNA. For example, the packaging limit for AAV is about 4.7 kb. The length of a single U6-sgRNA (+ restriction site for cloning) is 361 bp. Thus, one of ordinary skill in the art can easily accommodate approximately 12-16, eg, 13 U6-sgRNA cassettes in a single vector, which is the Golden Gate strategy (http://www) used for TALE assembly. It can be assembled by any suitable means, such as .genome-enginering.org/taleffectors /). Those skilled in the art will also increase the number of U6-sgRNA by about 1.5-fold, eg, 12-16, Tandem-guided strategies can also be used to increase, for example, from 13 to about 18-24, eg, about 19 U6-sgRNAs. Thus, one of ordinary skill in the art can use about 18-24 in a single vector, eg, AAV vector. A single promoter-RNA, such as about 19 promoter-RNAs, such as U6-sgRNA, can be readily reached. Further means of increasing the number of promoters and RNAs, such as one or more sgRNAs, in the vector is single. A promoter (eg, U6) is used to express an array of RNA, eg, sgRNA, separated by a cleavable sequence, and yet another means of increasing the number of promoter-RNA, eg, sgRNA, in the vector. Is to express an array of promoter-RNAs, such as sgRNAs, separated by a sequence that can be cleaved in the coding sequence or intron of the gene; and in this case it is advantageous to use the polymerase II promoter. Can increase expression and allow transcription of long RNAs in a tissue-specific manner (eg, http: //nar.oxfordjournals.org/content/34/7/e53.short, http: //www.nature .Com / mt / journal / v16 / n9 / abs / mt2008144a. Html). In an advantageous embodiment, the AAV targets up to about 50 genes. 6 Tandem sgRNAs can be packaged. Thus, from knowledge in the art and the teachings of the present disclosure, one of ordinary skill in the art will have multiple RNAs or guides or sgRNAs under the control of one or more promoters or operably or functionally linked to them. One or more vectors of expression, such as single vectors, can be readily prepared and used without any undue experimentation (especially with respect to the number of RNAs or guides or sgRNAs discussed herein).

The coding sequence and / or Cas coding sequence of one or more guide RNAs, eg, one or more sgRNAs, may be operably or operably linked to one or more regulatory elements, and thus this one. One or more regulatory elements drive expression. One or more promoters may be constitutive promoters and / or conditional promoters and / or inducible promoters and / or tissue-specific promoters. The promoters are RNA polymerase, pol I, pol II, pol III, T7, U6, H1, retrovirus Raus sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β- It can be selected from the group consisting of the actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. An advantageous promoter is the promoter U6.

Vector delivery Vector delivery, eg, plasmid, viral delivery: CRISPR enzyme, eg Cas9, and / or any book RNA, eg, guide RNA, with any suitable vector, eg, plasmid or viral vector, eg, adeno-associated virus. It can be delivered using viral (AAV), lentivirus, adenovirus, or viral vectors of other plasmids, or combinations thereof. Cas9 and one or more guide RNAs can be packaged in one or more vectors, such as plasmids or viral vectors. In some embodiments, the vector, eg, a plasmid or viral vector, is delivered to the tissue of interest, eg, by intramuscular injection, otherwise delivery is intravenous, transdermal, intranasal, oral, mucosal. Or by other delivery methods. Such delivery can be a single dose or multiple doses. For those skilled in the art, the actual dose delivered herein will be a variety of factors, such as vector selection, target cells, organisms, or tissues, general health of the subject to be treated, required transformation / It will be appreciated that it can vary significantly depending on the degree of modification, route of administration, method of administration, type of transformation / modification required, and the like.

Such doses may be, for example, carriers (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluents, pharmaceutically acceptable. It may further comprise a carrier (eg, phosphate buffered saline), a pharmaceutically acceptable excipient, and / or other compounds known in the art. Dosages include one or more pharmaceutically acceptable salts such as mineral salts such as hydrochlorides, hydrobromates, phosphates, sulfates; and organic acid salts such as acetates. It may further include propionate, malonate, benzoate and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gels or gelling substances, fragrances, colorants, microspheres, polymers, suspending agents and the like may also be present therein. In addition, one or more other conventional pharmaceutical ingredients include, for example, preservatives, wetting agents, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemistry. Stabilizers and the like may also be present, especially if the dosage form is in a reconstitutable form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethyl cellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, paraben, ethylvanillin, glycerin, phenol, Examples include parachlorophenol, gelatin, albumin, and combinations thereof. A thorough discussion of pharmaceutically acceptable excipients is available at REMINGTON'S PHARMACEUTICAL SCIENCES (Mac Pub. Co., N.J. 1991), which is incorporated herein by reference.

In one embodiment herein, delivered by adenovirus, the delivery can be a single booster dose comprising an adenoviral vector of at least 1 × 10 ⁵ particles (particle units, also called pu). In one embodiment of the specification, this dose is preferably at least about 1 × 10 ⁶ particles (eg, about 1 × 10 ⁶ to 1 × 10 ¹² particles), more preferably at least about 1 × 10 ^7. particles, more preferably at least about 1 × 10 ⁸ particles (e.g., particles of about 1 × ¹⁰ 8 to 1 × ^{10 11} particles, or about 1 × ¹⁰ 8 to 1 × ^{10 12),} and most preferably at least about 1 × 10 ⁰ particle (e.g., about 1 × ¹⁰ 9 ~1 × ^{10 10} particles of the particle or about 1 × ¹⁰ 9 ~1 × ^{10 12),} or even at least about 1 × ^{10 10} particles (e.g., about 1 × 10 ¹⁰ to 1 × 10 ¹² particles) adenovirus vector. Alternatively, the dose is about 1 × 10 ¹⁴ or less particles, preferably about 1 × 10 ¹³ or less particles, even more preferably about 1 × 10 ¹² or less particles, even more preferably about 1 × 10 ¹¹ or less. Particles, and most preferably particles of about 1 × 10 ¹⁰ or less (eg, particles of about 1 × 10 ⁹ or less). Thus, the doses are, for example, about 1 × 10 ⁶ particle units (pu), about 2 × 10 ⁶ pu, about 4 × 10 ⁶ pu, about 1 × 10 ⁷ pu, about 2 × 10 ⁷ pu, about 4 ×. 10 ⁷ pu, about 1 x 10 ⁸ pu, about 2 x 10 ⁸ pu, about 4 x 10 ⁸ pu, about 1 x 10 ⁹ pu, about 2 x 10 ⁹ pu, about 4 x 10 ⁹ pu, about 1 x 10 ¹⁰ pu, about 2 x 10 ¹⁰ pu, about 4 x 10 ¹⁰ pu, about 1 x 10 ¹¹ pu, about 2 x 10 ¹¹ pu, about 4 x 10 ¹¹ pu, about 1 x 10 ¹² pu, about 2 x 10 A single dose of an adenovirus vector containing an adenovirus vector of ¹² pu, or about 4 × 10 ¹² pu, may be included. See, for example, the adenovirus vector of US Pat. No. 8,454,972 B2, granted to Nabel et al. On June 4, 2013, which is incorporated herein by reference; the dosage is in that paragraph. See lines 36-58 of 29. In one embodiment of the specification, the adenovirus is delivered by multiple doses.

In one embodiment of the specification, delivery is by AAV. A therapeutically effective amount of AAV for in vivo delivery to humans is considered to be in the range of about 20 to about 50 ml saline containing about 1 x 10 ¹⁰ to about 1 x 10 ¹⁰ functional AAV / ml solutions. Be done. The dose can be adjusted to balance the therapeutic effect against any side effects. In one embodiment of the specification, the dose of AAV is generally about 1 × 10 ⁵ to 1 × 10 ⁵⁰ genomic AAV, about 1 × 10 ⁸ to 1 × 10 ²⁰ genomic AAV, about 1 × 10 ¹⁰ to about. Concentration ranges of 1 × 10 ¹⁶ genomes, or about 1 × 10 ¹¹ to about 1 × 10 ¹⁶ genomes AAV. The human dose can be about 1 × 10 ¹³ genomic AAV. Such concentrations can be delivered in about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution. Other effective doses can be readily established by one of ordinary skill in the art by routine testing to establish a dose-response curve. For example, Hajjar, et al. Granted on March 26, 2013. U.S. Pat. No. 8,404,658 B2, column 27, lines 45-60.

Packaging and Promoter Overview Methods for packaging Cas9-encoding nucleic acid molecules, such as DNA, into vectors, such as viral vectors, to mediate genomic modification in vivo include:
To achieve NHEJ-mediated gene knockout:
Single viral vector:
A vector containing two or more expression cassettes:
-Promoter-Cas9 coding nucleic acid molecule-Terminator-Promoter-gRNA1-Terminator-Promoter-gRNA2-Terminator-Promoter-gRNA (N) -Terminator (up to vector size limit)
Double viral vector:
Vector 1 containing one expression cassette to drive Cas9 expression
-Promoter-Cas9 coding nucleic acid molecule-Terminator-Vector 2 containing another expression cassette for driving the expression of one or more guide RNAs
-Promoter-gRNA1-terminator-Promoter-gRNA (N) -terminator (up to the size limit of the vector)

To mediate homology-dependent repair • In addition to the single and double viral vector techniques described above, additional vectors can be used to deliver homology-dependent repair templates.

Promoters used to drive Cas9-encoding nucleic acid molecule expression can include:
AAV ITR can serve as a promoter: this is advantageous because it eliminates the need for additional promoter elements (which can take up space in the vector). The extra space available can be used to drive the expression of additional elements (such as gRNA). Also, since ITR activity is relatively weak, it can be used to reduce the potential toxicity due to overexpression of Cas9.
-For ubiquitous expression, promoters: CMV, CAG, CBh, PGK, SV40, ferritin heavy chain or light chain, etc. can be used.-For expression in the brain or other CNS, promoter: for all neurons Synapsin I, CaMKII alpha for excitatory neurons, GAD67 or GAD65 for GABAergic neurons, VGAT, etc. can be used. • An albumin promoter can be used for expression in the liver.
-SP-B can be used for expression in the lung.
-ICAM can be used for endothelial cells.
-IFNβ or CD45 can be used for hematopoietic cells.
-OG-2 can be used for osteoblasts.

Promoters used to drive guide RNAs can include:
-Use of Pol II promoter and intron cassette to express Pol III promoter gRNA such as U6 or H1

Crystallization and Structure of CRISPR-Cas9 Crystallization and Crystal Structure of CRISPR-Cas9 Crystallization can be obtained by protein crystallographic techniques including batch, liquid cross-linking, dialysis, vapor diffusion, and water droplets. Generally, crystals are obtained by dissolving substantially pure CRISPR-Cas9 and the nucleic acid molecule to which the CRISPR-Cas9 binds in an aqueous buffer containing a precipitant at a concentration slightly lower than the concentration required for precipitation. Grow. Water is removed by controlled evaporation to create precipitation conditions and this is maintained until crystal growth ceases. See Nishimasu et al.

Use of Crystal, Crystal Structure, and Atomic Structure Coordinates: Crystals, and especially the atomic structure coordinates obtained from crystals, have a wide range of uses. Crystalline and structural coordinates are particularly useful for identifying compounds that bind to CRISPR-Cas9 (nucleic acid molecules) and CRISPR-Cas9 that can bind to specific compounds (nucleic acid molecules). Therefore, the structural coordinates described herein can be used as a phase model in determining the crystal structure of additional synthetic or mutated CRISPR-Cas9, Cas9, nickase, binding domains. By providing the crystal structure of CRISPR-Cas9 complexed with a nucleic acid molecule, one of ordinary skill in the art can gain insight into CRISPR-Cas9. This insight provides a means of designing modified CRISPR-Cas9, such as by adding functional groups such as repressors or activators. Functional groups such as repressors or activators can be added to the N-terminus or C-terminus of CRISPR-Cas9, but the crystal structure appears to have the N-terminus covered or hidden, while the C-terminus It has been shown to be more accessible for functional groups such as repressors or activators. In addition, the crystal structure has a mobile loop suitable for adding functional groups such as activators or repressors between CRISPR-Cas9 (Streptococcus pyogenes) residues of about 534-676. Indicates that it exists. Addition can be via a linker, such as mobile glycine-serine (GlyGlyGlySer) or (GGGS) 3, or a robust α-helix linker, such as (Ala (GluAlaAlaAlaLys) Ala). In addition to the mobile loop, there are also nuclease or H3, H2, and helical regions. "Helix" or "helical" means a helix known in the art, including, but not limited to, an α-helix. In addition, the term helix or helical is also used to refer to a c-terminal helical element with an N-terminal turn.

The provision of the crystal structure of CRISPR-Cas9 complexed with a nucleic acid molecule enables novel drug or compound drug discovery, identification, and design techniques for compounds capable of binding to CRISPR-Cas9, and thus the present disclosure is described. Multicellular organisms such as algae, plants, invertebrates, fish, amphibians, reptiles, birds, mammals; for example, cultivated plants, domestic animals (eg, producing animals such as pigs, cows, chickens; pet animals, for example cats , Dogs, rodents (rabbits, snails, hamsters); experimental animals such as mice, rats), and tools useful for diagnosing, treating, or preventing pathologies or disorders in humans. Therefore, the present disclosure provides a computer-based method for rationally designing the CRISPR-Cas9 complex. This rational design is: part or all of the crystal structure table and / or the figure relating to the crystal structure (eg, at least two or more of the structures, eg, at least 5, preferably at least 10, and more preferably at least. A step of providing the structure of the CRISPR-Cas9 complex as defined by the coordinates of 50, and even more preferably at least 100 atoms); Nishimasu et al. To provide the structure of the desired nucleic acid molecule as to which CRISPR-Cas9 complex is desirable; and to the structure of the CRISPR-Cas9 complex as defined by some or all coordinates to the desired nucleic acid molecule. A fitting step, wherein the fitting is defined by some or all coordinates of the desired nucleic acid molecule to bind to one or more CRISPR-Cas9 complexes involved in the desired nucleic acid molecule. It may include steps that include achieving a putative modification of one or more CRISPR-Cas9 complexes as per. This method or fitting of this method comprises at least two or more active sites or binding regions (eg, at least 5, at least 5, preferably at least 10, more preferably at least 50, and even more advantageously. To model the neighborhood of at least 100 atoms), use the desired atomic coordinates of the CRISPR-Cas9 complex as defined by some or all coordinates in the vicinity of the active site or binding region. Can be done. These coordinates can be used to define a space for later screening "in silico" for desired or candidate nucleic acid molecules. Therefore, the present disclosure provides a computer-based method for rationally designing the CRISPR-Cas9 complex. This method: provides the coordinates of at least two atoms in the crystal structure table (“selected coordinates”); Nishimasu et al. Can include the step of providing the structure of the candidate or desired nucleic acid molecule; and the step of fitting the candidate structure to the selected coordinates. In this way, one of ordinary skill in the art can also fit functional groups and candidate or desired nucleic acid molecules. For example, the coordinates of some or all (eg, at least 2 or more of the structure, eg, at least 5, preferably at least 10, more preferably at least 50, and even more preferably at least 100 atoms). Steps to provide the structure of the CRISPR-Cas9 complex as defined by; Steps to provide the structure of the desired nucleic acid molecule as to which CRISPR-Cas9 complex is desirable; in the crystal structure table and / or in the figure relating to the crystal structure. The structure of the CRISPR-Cas9 complex as defined by some or all of the coordinates of; Nishimasu et al. The step of fitting to a desired nucleic acid molecule, wherein the fitting defines the desired nucleic acid molecule so as to bind to one or more CRISPR-Cas9 complexes involved in the desired nucleic acid molecule. Steps involving achieving putative modifications of one or more CRISPR-Cas9 complexes as is; selecting one or more putative matching CRISPR-Cas9-desired nucleic acid molecule complexes Steps to, such one or more putatively compatible CRISPR-Cas9-desired nucleic acid molecule complexes, eg, in a location (eg, in a mobile loop) where a functional group (eg, activator, repressor) is located. (Position of) and / or fitting to a functional group with respect to a putative modification of one or more putatively compatible CRISPR-Cas9-desired nucleic acid molecule complexes to create a location for the functional group. Steps to do.

However, information on the SpCas9 crystal structure (see Nishimasu et al.) Is that the off-target effect achieved by the mutations of the invention as disclosed herein is reduced; or certain mutations are off-target effects. It was not possible to predict that a decline in However, given that the disclosure now provides information on mutations that result in or achieve reduced off-target effects, those skilled in the art can readily apply the teachings herein in conjunction with information on SpCas9 crystal structure and Cas9 sequences. By performing sequence and structural analysis across the entire Cas9, similar amino acids that can be mutated or modified in a manner similar to those herein are determined, and mutations or modifications result in reduced off-target effects. Mutant or modified Cas9 can be obtained.

Accordingly, the present disclosure, along with information on SpCas9 crystals, is in the vicinity of one or more mutations or one or more modifications disclosed herein, or such one or more mutations or one or more. Coordinates located in the active site or binding region located in close proximity to multiple modifications can be used; and therefore further mutations or modifications of SpCas9 or similar mutations or modifications in the Cas9 ortholog. The method of determination may use consideration, eg, comparative consideration of one or more subdomains of interest in the CRISPR-Cas9 complex. Methods for determining further mutations or modifications of SpCas9 or similar mutations or modifications in the Cas9 ortholog can be performed using domain or subdomain coordinates. The method optionally synthesizes a candidate or desired nucleic acid molecule and / or CRISPR-Cas9 system from "in silico" output, and "wet" or actual mutation or modification binding and / or activity and / Or may include a step of testing a reduction in off-target effect. The CRISPR-Cas9 system, including mutations or modifications, may optionally contain functional groups. These methods may include observing cells or organisms containing cells with respect to reduction of symptoms or pathology or disease, including the desired response, eg, reducing off-target effects. The steps provided in the structure of a candidate nucleic acid molecule may include selecting compounds by computerally screening a database containing nucleic acid molecule data, such as such data on pathology or disease. From the structural and chemical properties of the CRISPR-Cas9 complex from its crystal structure or its domain or region, the 3D descriptor for binding of the candidate nucleic acid molecule, taking into account mutations or modifications as disclosed herein. It can be derived from the derived geometric and functional constraints. In essence, this descriptor can be a type of virtual modification of the CRISPR-Cas9 complex crystal structure herein for CRISPR-Cas9 to bind to a candidate or desired nucleic acid molecule. The "wet" step herein can then be performed using a descriptor and a nucleic acid molecule with presumably good binding.

"Fiting" determines the interaction between at least one atom of a candidate and at least one atom of the CRISPR-Cas9 complex by automatic or semi-automatic means, and how stable such interaction is Can mean to calculate. The interaction may include attractive and repulsive forces generated by electric charges and steric factors. A "subdomain" can mean at least one of the secondary structures, eg, 1, 2, 3, or four complete elements. Specific regions or domains of CRISPR-Cas9 include those identified in the crystal structure table and the corresponding figures; see Nishimasu et al.

In any case, the location of the mutation / modification identified herein can be applied to the Cas9 ortholog based on sequence and structural position comparisons using the crystal structure of the CRISPR-SpCas9 complex, and thus CRISPR-. The three-dimensional structure of the Cas9 (eg, S. pyogenes Cas9; see Nishimasu et al) complex provides additional tools for identifying further mutations in the Cas9 ortholog in the context of the present invention. provide. Crystal structures also include novel specific Cas9s, such as those having one or more mutations or one or more modifications herein, and any one or more of various functional groups, eg, Includes, has, or is linked to transcriptional repressors, transcriptional activators, nuclease domains, DNA methyltransferases, protein acetyltransferases, protein deacetylases, protein methyltransferases, protein deaminase, protein kinases, and protein phosphatases as fusion partners. It can also be the basis for the design of things; and in some embodiments, the functional domain is a posterior regulator; eg, Zhang et al. , U.S. Pat. No. 8,507,272, again, and all citations and all application citations herein are hereby by reference. (It is mentioned that it is incorporated), with a modification of Cas9, with a novel nickase). From this disclosure and information on the three-dimensional structure of the CRISPR-Cas9 (Streptococcus pyogenes Cas9) crystal structure, a computer modeling program can be used to create a binding site or CRISPR-Cas9 system (eg, Streptococcus pyogenes (S. pyogenes)). Various molecules that are expected to interact with potential or identified sites, such as other structural or functional features of pyogenes) Cas9), can be designed or identified. Potentially bound compounds (“binders”) can be investigated using computer modeling using a docking program. Docking programs are known; for example, GRAM, DOCK or AUTODOCK (Walters et al. Drag Discovery Today, vol. 3, no. 4 (1998), 160-178, and Dunblack et al. Folding and Design 2 (1997), See 27-42). In this procedure, how well the potential binder shape and chemical structure binds to the CRISPR-Cas9 system (eg, S. pyogenes Cas9) by computer fitting of the potential binder. May include making sure. The active or binding sites of the CRISPR-Cas9 system (eg, S. pyogenes Cas9) may be manually examined with computer assistance. Programs such as GRID (P. Goodford, J. Med. Chem, 1985, 28, 849-57) -a program that determines the likely sites of interaction between molecules with various functional groups-also bind. It can be used in the analysis of active or binding sites to predict the partial structure of a compound. Using a computer program, two binding partners, such as the CRISPR-Cas9 system (eg, S. pyogenes Cas9) and the candidate nucleic acid molecule or the nucleic acid molecule and the candidate CRISPR-Cas9 system (eg, S. pyogenes) ) Cas9) attraction, repulsion or steric hindrance can be estimated; and with the CRISPR-Cas9 crystal structure (S. pyogenes Cas9) according to this specification, such a method is possible. In general, the tighter the fit, the less steric hindrance and the greater the attractive force, the stronger the potential binder, because these properties are consistent with the tighter binding constants. Furthermore, the higher the specificity in the design of the candidate CRISPR-Cas9 system (eg, S. pyogenes Cas9), the more likely it is that it will not further interact with off-target molecules. The "wet" method is also possible by the present invention. For example, in some embodiments, the present disclosure is a binder for a candidate CRISPR-Cas9 system (eg, S. pyogenes Cas9) that has bound to a candidate CRISPR-Cas9 system (eg, S. pyogenes Cas9). A method for determining the structure of (eg, a target nucleic acid molecule) is provided, wherein the method is (a) the first crystal or candidate of the candidate CRISPR-Cas9 system (S. pyogenes Cas9) according to the present disclosure. Steps to provide a second crystal of the CRISPR-Cas9 system (eg, S. pyogenes Cas9), (b) binding the first or second crystal under conditions where a complex can be formed. Steps of contact with the agent; and (c) of the candidate (eg, CRISPR-Cas9 series (eg, S. pyogenes Cas9) or CRISPR-Cas9 series (S. pyogenes Cas9) complex A second crystal may have essentially the same coordinates as discussed herein, but with slight changes in the CRISPR-Cas9 system (eg, for example, Streptococcus pyogenes (S. streptococcus). Changes from Cas9 in such a system (for example, Streptococcus pyogenes (S. pyogenes) Cas9), where "for example, S. pyogenes Cas9", this Cas9 is Cas9. (Indicating that it can be or can be derived from S. pyogenes or its ortholog), the crystals can be formed in different spatial groups. The present disclosure presents in lieu of or in addition to the "Incilico" method, a binding agent (eg, a target nucleic acid molecule) and a candidate CRISPR-Cas9 system (eg, S. pyogenes) Cas9 for selecting compounds with binding activity. ), Or a candidate binding agent (eg, target nucleic acid molecule) and a CRISPR-Cas9 system (eg, S. pyogenes Cas9), or a candidate binding agent (eg, target nucleic acid molecule) and a candidate CRISPR-Cas9 system (eg, Streptococcus pyogenes). Further includes other "wet" methods, including high throughput screening of S. pyogenes Cas9) (one or more CRISPR-Cas9 systems described above may or may not contain one or more functional groups). .. A pair of binder and CRISPR-Cas9 system exhibiting binding activity is selected and further crystallized in the CRISPR-Cas9 crystal having the structure of the present specification for X-ray analysis, for example by co-crystallization or immersion. be able to. Design possible pairs of binders and CRISPR-Cas9 systems by determining preferred fitting properties, eg, those having the expected strong attractive force based on the pair of binder and CRISPR-Cas9 crystal structure data herein. Once identified or selected, these possible pairs can then be screened for activity by the "wet" method. As a result, in one aspect, the invention: the step of obtaining or synthesizing a possible pair; and a binding agent (eg, a target nucleic acid molecule) and a candidate CRISPR-Cas9 system (eg, S. pyogenes Cas9), or Candidate binding agent (eg, target nucleic acid molecule) and CRISPR-Cas9 system (eg, S. pyogenes Cas9), or candidate binding agent (eg, target nucleic acid molecule) and candidate CRISPR-Cas9 series (eg, Streptococcus pyogenes (S)) .Pyogenes) Cas9) (the one or more CRISPR-Cas9 systems described above may or may not contain one or more functional groups) may include the step of contacting them to determine their binding capacity. In the latter step, this contact is advantageously a function-determining condition. In lieu of or in addition to performing such an assay, the present disclosure: the steps of obtaining or synthesizing one or more complexes from said contact, and for example X-ray diffraction or NMR or the like of the one or more complexes. It may include the step of analyzing by the means of the above to determine the binding or interacting ability. Next, detailed structural information regarding the binding can be obtained, and the structure or function of the candidate CRISPR-Cas9 system or its constituents can be adjusted based on this information. These steps may be repeated and repeated as needed. Alternatively or additionally, potential CRISPR-Cas9 systems from or in the methods described above confirm their function, including whether they provide the desired results (eg, symptom relief, treatment). It may be with nucleic acid molecules in vivo, including, but not limited to, by administration to living organisms (including non-human animals and humans) to prove or prove.

The present disclosure is unknown by using the structural coordinates of the literature discussed herein, in particular when adjusted for one or more modifications or mutations discussed herein. Further included are methods of determining the three-dimensional structure of one or more CRISPR-cas systems or complexes of the structure. For example, when X-ray crystallographic data or NMR spectroscopic analysis data is provided for a CRISPR-Cas system or complex with an unknown crystal structure, by interpreting the data using the structure of the CRISPR-Cas9 complex. , Techniques such as phase modeling in the case of X-ray crystallography may provide expected structures of unknown systems or complexes. Thus, the method is: Representation of a CRISPR-Cas system or complex with an unknown crystal structure with a similar representation of the CRISPR-Cas 9 system or complex of the crystal structure cited herein (advantageously homologous or homologous). Steps to align with one or more modifications or one or more mutations herein to match similar regions (eg, homologous or similar sequences); CRISPR of unknown crystal structure -Steps to model the structure of a matched homologous or similar region (eg, sequence) of a Cas system or complex; and conformation for an unknown crystal structure that substantially maintains the structure of the matched homologous region. For example, favorable interactions may include determining steps (given that low energy conformations should be formed). “Homologous region” refers, for example, with respect to amino acids, for example, amino acid residues in two sequences having the same or similar aliphatic, aromatic, polar, negative or positive charge, side chain chemical groups. Homological regions for nucleic acid molecules are at least 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%. , Or 96%, or 97%, or 98%, or 99% homology or identity. The same and similar regions are sometimes described by those skilled in the art as "invariant" and "conserved," respectively. Advantageously, the first and third steps are performed by computer modeling. Homology modeling is a technique well known to those of skill in the art (see, eg, Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513). The computer representation of the storage region of the CRISPR-Cas9 crystal structure and the computer representation of the CRISPR-Cas system of unknown crystal structure in the present specification are useful for predicting and determining the crystal structure of the CRISPR-Cas system of unknown crystal structure. Furthermore, aspects of the invention utilizing the CRISPR-Cas9 crystal structure in in silico apply equally to the novel one or more mutations or one or more modifications and the CRISPR-Cas crystal structure herein. obtain. In this way, a library of CRISPR-Cas crystal structures can be obtained. Therefore, the present disclosure provides a rational CRISPR-Cas system design. For example, if the conformation or crystal structure of a CRISPR-Cas system or complex is determined by the methods described herein, including utilizing knowledge of the art from the references cited herein. Such conformations can be used in the computer-based manner herein to determine the conformation or crystal structure of other CRISPR-Cas systems or complexes whose crystal structure is still unknown. All of these crystal structure data may be in a database, and the methods herein involve the crystal structure of this specification or a portion thereof compared to one or more crystal structures in a library. Can be more robust by comparing. The present disclosure further includes systems such as computer systems intended to generate structures of CRISPR-cas systems or complexes and / or to carry out rational design thereof. This system includes: atomic coordinate data or data derived from it, for example by modeling, which is the three-dimensional structure of the CRISPR-cas system or complex or at least one domain or subdomain thereof, or its structural factor data. By definition, the structural factor data can be derived from atomic coordinate data. The disclosure also includes a computer-readable medium containing atomic coordinate data, which defines a three-dimensional structure of a CRISPR-cas system or complex or at least one domain or subdomain thereof, or structural factor data thereof. "Computer readable medium" refers to any medium that can be read and accessed directly by a computer, including but not limited to: magnetic storage media; optical storage media; electrical storage media; cloud storage devices, and hybrids of these categories. included. By providing such a computer-readable medium, atomic coordinate data can be routinely accessed for modeling or other "in silico" methods. The disclosure further includes methods of doing business by providing access to such computer-readable media, eg, by subscription fee method, via the Internet or global communications / computer networks; or computer systems by the user by subscription fee method. It may be available. "Computer system" refers to hardware means, software means, and data storage means used in the analysis of atomic coordinate data of the present invention. Minimal hardware means of the computer-based system of the present invention may include a central processing unit (CPU), input means, output means, and data storage means. Desirably, a display or monitor is provided to visualize the structural data. The present disclosure presents the information herein or any method described herein or information obtained in steps thereof, such as by telecommunications, telephone, media, mass media, presentations, the Internet, e-mail, etc. Further includes methods of communication. The crystal structure of the present disclosure can be analyzed to create one or more Fourier electron density diagrams of the CRISPR-cas system or complex. The Fourier electron density diagram can be calculated based on the X-ray diffraction pattern. These figures can then be used to characterize binding or other interactions. The electron density diagram is calculated by using a program of the CCP4 computer package (Collaboration Computing Project, No. 4. The CCP4 Suite: Programs for Protein Crystallogphy, Acta Crystallogratica, D50, 1994, 760-763, etc.). Can be done. Programs such as "QUANTA" (1994, San Diego, Calif .: Molecular Simulations, Jones et al., Acta Crystallography A47 (1991), 110-119) can be used for diagram visualization and model construction.

The crystal structure referred to herein provides atomic coordinate data for CRISPR-Cas9 (S. pyogenes), and each atom is listed with the following unique number; for each amino acid residue. Crystal axis of chemical element and its position (determined by electron density chart and antibody sequence comparison), amino acid residue where the element is located, chain identification name, number of residues, atomic position (Angstrom unit) of each atom Coordinates defined for (eg, X, Y, Z), atom occupancy at each position, "B", isotropic displacement parameter ( ² units of angstrom) that describes the movement of the atom around the atomic center, And atomic number.

In certain embodiments of the invention, variations in conformation in the crystal structure of the CRISPR-Cas9 system or components of the CRISPR-Cas9 system may be important for the function of the CRISPR-Cas system, nucleotide (RNA or DNA) structural regions. Provides important and definitive information about the mobility or migration of protein structural regions to. Cas9 as a CRISPR enzyme in the present application (eg, S. pyogenes Cas9: "Crystal structure of cas9 in complex with guide RNA and gene". Nishimasu, H., Ran, FA., Hsu, PD., Konermann, S., Shehata, SI., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell Feb 27. 2014) .156 (5): 935-49; or Sa Cas9: "Crystal Streptococcus aureus Cas9", Nishimasu et al., Cell 162, 1113-1126. The CRISPR-Cas system may be further engineered and optimized using the structural information provided for 2015)), and can be estimated from this to further investigate the structure-functional relationship in other CRISPR enzyme systems. One aspect of the present invention relates to the crystal structure of S. pyogenes Cas9, which has formed a complex with sgRNA and its target DNA at 2.4 Å resolution. From this structure, a two-lobe structure composed of a target recognition lobe and a nuclease lobe has been clarified, and an sgRNA: DNA duplex is accepted in a positively charged groove at the interface between them. The recognition lobe is essential for the binding of sgRNA to DNA, and the nuclease lobe contains the HNH and RuvC nuclease domains, which are in appropriate positions for cleavage of the complementary and non-complementary strands of the target DNA, respectively. This high-resolution structural and functional analysis provided herein elucidates the molecular mechanism of RNA-guided DNA targeting by Cas9 and provides sufficient information to create an optimized CRISPR-Cas system and its constituents. To do.

In a detailed embodiment of the invention, the crystal structure provides an important step towards understanding the molecular mechanism of RNA-guided DNA targeting by Cas9. The structural and functional analysis herein provides a useful foothold for the rational engineering of Cas9-based genomic regulatory techniques, and Cas9-mediated PAM sequence recognition on target DNA or mismatch between sgRNA: DNA duplexes. Can provide guidance on tolerance. Aspects of the invention also relate to truncation mutations, eg, S. pyogenes Cas9 truncation mutations can facilitate the packaging of Cas9 into size-restricted viral vectors for in vivo therapeutic applications. .. Similarly, by engineering the PAM Interaction (PI) domain, it may be possible to program PAM specificity, improve target site recognition fidelity, and increase the versatility of the Cas9 genomic engineering platform. .. In addition, by engineering the PAM Interaction (PI) domain, it is possible to program PAM specificity, improve the fidelity of target site recognition, and increase the versatility of Cas, eg Cas9 genome engineering platforms. Can be. Cas proteins such as the Cas9 protein are described, for example, by Kleinstiber BP et al. "Engineered CRISPR-Cas9 nucleoses with alternate PAM Specificities" with altered PAM specificity. Nature. 2015 Jul 23; 523 (7561): 481-5. It can be engineered to alter its PAM specificity as described in doi: 10.1038 / nature14592.

The present invention includes an optimized functional CRISPR-Cas enzyme system. Specifically, this CRISPR enzyme comprises one or more mutations that convert a functional domain exhibiting a function of interest into a DNA-binding protein to which it can be recruited, added, inserted or bound. In certain embodiments, the CRISPR enzyme is one or more, including, but not limited to, D10A, E762A, H840A, N854A, N863A or D986A (based on the amino acid position numbering of S. pyogenes Cas9). And / or one or more mutations are in the RuvC1 or HNH domain of the CRISPR enzyme, or are other mutations as discussed herein. In some embodiments, the CRISPR enzyme has one or more mutations in the catalytic domain, where when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence is CRISPR to the target sequence. It leads to sequence-specific binding of the complex, where the enzyme further comprises a functional domain.

The structural information provided herein allows the sgRNA (or sgRNA) with a target DNA and CRISPR enzyme (eg, Cas9) to allow engineering or alteration of the sgRNA structure to optimize the function of the entire CRISPR-Cas system. It is possible to investigate chimeric RNA) interactions. For example, one or more individual RNA loops or one or more individual RNA loops or one or more individual RNA loops capable of recruiting an adapter protein capable of binding to individual one or more sequences. Insertion of a distinct sequence may extend the loop of the sgRNA without collision with the Cas9 protein.

Functional Variants of Enzymes of the Invention In embodiments, Cas9 proteins as referred to herein also include functional variants. A "functional variant" of a protein, as used herein, refers to a variant of such protein that retains at least partially the activity of that protein. Functional variants can include mutants, including polymorphisms, which can be insertion, deletion, or substitution mutants. Also included within the functional variants are fusion products of such proteins with other, usually unrelated nucleic acids, proteins, polypeptides or peptides. The functional variant may be naturally occurring or artificial. Advantageous embodiments may include engineered or non-naturally occurring type II RNA targeting effector proteins such as Cas9 or its orthologs or homologs.

General Information Regarding Protein Mutation According to the Invention The present invention includes a CRISPR Cas complex that includes a CRISPR enzyme and a guide RNA (sgRNA), where the CRISPR enzyme is at least one mutation (hence the CRISPR enzyme is Contains less than 5% nuclease activity of CRISPR enzymes without at least one mutation), and optionally at least one or more nuclear localization sequences; guide RNAs (sgRNAs) are the genome of interest of the cell. Contains a guide sequence capable of hybridizing to a target sequence at the locus; and here: the CRISPR enzyme associates with more than one functional domain; or at least one loop of sgRNA binds to one or more adapter proteins Modified by the insertion of one or more individual RNA sequences, where the adapter protein associates with more than one functional domain; or the CRISPR enzyme associates with one or more functional domains, and of the sgRNA. At least one loop is modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins, where the adapter proteins associate with one or more functional domains.

Functional Domains and Adapter Proteins; Aptamers Adapter proteins can include, but are not limited to, orthogonal RNA-binding protein / aptamer combinations that fall within the diversity of bacteriophage coat proteins. The list of such coated proteins includes, but is not limited to: Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. These adapter proteins or orthogonal RNA-binding proteins can further recruit effector proteins or fusions containing one or more functional domains. In some embodiments, the functional domains are transposase domain, integrase domain, recombinase domain, resolverase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, deaminase, histone acetylase. Lase domain, histone deacetylase domain, nuclease domain, repressor domain, activator domain, nuclear localization signal domain, transcriptional regulatory protein (or transcription complex recruitment) domain, cell uptake activity-related domain, nucleic acid binding domain, antibody Presentation domain, histone modifiers, histone modifier recruiters; histone modifier inhibitors, histone methyltransferases, histone demethylases, histone kinases, histone phosphatases, histone ribosyrase, histone deribosylase, histone ubiquitinase, histones It can be selected from the group consisting of duubiquitinase, histone biotinase and histone tail protease.

In some preferred embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is a SID, or a concatemer of the SID (eg, SID4X). In some embodiments, the functional domain is an epigenetic modified domain, providing an epigenetic modified enzyme. In some embodiments, the functional domain is the activation domain, which may be the P65 activation domain. In some embodiments, the functional domain is a deaminase such as cytidine deaminase. The cytidine deaminase can be directed to the target nucleic acid, where it leads to the conversion of cytidine to uridine, resulting in a C to T substitution (G to A on the complementary strand). In such an embodiment, nucleotide substitutions can be achieved without DNA cleavage.

In one aspect, surveyor analysis is used to identify indel activity / nuclease activity. Survey analysis generally includes extraction of genomic DNA, PCR amplification of the genomic region flanking the CRISPR target site, purification of the product, and reannealing to allow heteroduplex formation. After annealing, the product is treated with SURVEYOR nuclease and SURVEYOR Enhancer S (Transgenemics) according to the manufacturer's recommended protocol. The analysis can be performed on a polyacrylamide gel by a known method. Quantification can be based on relative band intensities.

*** Inducible enzyme and split enzyme ("Split-Cas9")
In some embodiments, the present invention
A first Cas9 fusion construct bound to the first half of the inducible dimer, and a second Cas9 fusion construct bound to the second half of the inducible dimer,
Provided a non-naturally occurring or engineered inducible Cas9 CRISPR-Cas system, including
The first Cas9 fusion construct is operably linked to one or more nuclear localization signals.
The second Cas9 fusion construct is operably linked to one or more extranuclear translocation signals.
Upon contact with the inducer energy source, the first and second halves of the inducible dimer become one.
When the first and second halves of the inducible dimer are integrated, the first and second Cas9 fusion constructs can constitute a functional Cas9 CRISPR-Cas system.
The Cas9 CRISPR-Cas system contains a guide RNA (gRNA) containing a guide sequence capable of hybridizing to the target sequence at the target genomic locus of the cell, and the functional Cas9 CRISPR-Cas system binds to the target sequence. And optionally, the genomic loci are edited to alter gene expression.

In some aspects of the invention, in the inducible Cas9 CRISPR-Cas system, the inducible dimer is, contains, or essentially consists of an inducible heterodimer. In some embodiments, in the inducible Cas9 CRISPR-Cas system, the first half or first portion or first fragment of the inducible heterodimer is FKBP, optionally FKBP12, or comprises it. Or consists of, or is essentially. In some aspects of the invention, in the inducible Cas9 CRISPR-Cas system, the second half or second portion or second fragment of the inducible heterodimer is the FRB or contains it. Or consists of, or is essentially. In some aspects of the invention, in the inducible Cas9 CRISPR-Cas system, the arrangement of the first Cas9 fusion construct is, or comprises, or consists of the N'terminal Cas9 part-FRB-NES. Or then it becomes essential. In some aspects of the invention, in the inducible Cas9 CRISPR-Cas system, the arrangement of the first Cas9 fusion construct is, or comprises, NES-N'terminal Cas9 part-FRB-NES. Or then become essential. In some aspects of the invention, in the inducible Cas9 CRISPR-Cas system, the arrangement of the second Cas9 fusion construct is, or comprises, or is essentially C'terminal Cas9 part-FKBP-NLS. Or consists of it. In some embodiments, the invention presents in an inducible Cas9 CRISPR-Cas system in which the arrangement of the second Cas9 fusion construct is, or includes, NLS-C'terminal Cas9 part-FKBP-NLS, or from it. Provide to be, or be essentially, to be. In some embodiments, the inducible Cas9 CRISPR-Cas system may have a linker that separates the Cas9 part from a half or portion or fragment of the inducible dimer. In some embodiments, in the inducible Cas9 CRISPR-Cas system, the inducer energy source is, or comprises, or consists essentially of rapamycin. In some embodiments, in the inducible Cas9 CRISPR-Cas system, the inducible dimer is an inducible homodimer. In some embodiments, in the inducible Cas9 CRISPR-Cas system, Cas9 is FnCas9. In some embodiments, in an inducible Cas9 CRISPR-Cas system, a functional domain comprising one or more functional domains, eg, optionally transcriptional activators, transcriptional or Fok1 nucleases, in one or both parts of Cas9. Are meeting. In some embodiments, in the inducible Cas9 CRISPR-Cas system, the functional Cas9 CRISPR-Cas system binds to the target sequence, and the enzyme is optionally at least 97 when compared to Cas9 without at least one mutation. Dead Cas9 with% or 100% reduction in nuclease activity (or less than 3% and preferably 0% nuclease activity). The present invention further includes polynucleotides encoding inducible Cas9 CRISPR-Cas systems as discussed herein, and certain aspects of the invention provide it.

In some embodiments, the invention binds to the first half or portion or fragment of an inducible dimer and acts on one or more nuclear localization signals as discussed herein. Provided is a vector for delivering a first Cas9 fusion construct that is ligated as possible. In some embodiments, the present invention is a second Cas9 fusion construct bound to a second half or portion or fragment of an inducible dimer and operably linked to one or more extranuclear translocation signals. A vector for delivering the above is provided.

In some embodiments, the invention is capable of binding to a first half or portion or fragment of an inducible dimer and acting on one or more nuclear localization signals, as discussed herein. A first Cas9 fusion construct linked to; and one or more nuclear localization signals that bind to a second half or portion or fragment of an inducible dimer as discussed herein. Provided is a vector for delivering both a second Cas9 fusion construct, operably linked to.

In some embodiments, the vector may be a single plasmid or an expression cassette.

The present invention is, in some embodiments, a eukaryotic host cell transformed with any of the vectors discussed herein, or expressing an inducible Cas9 CRISPR-Cas system as discussed herein. Provide a cell line.

The present invention presents, in some embodiments, transgenic organisms transformed with any of the vectors discussed herein, or expressing the inducible Cas9 CRISPR-Cas system discussed herein, or progeny thereof. provide. In some embodiments, the invention provides a model organism that constitutively expresses the inducible Cas9 CRISPR-Cas system as discussed herein.

In some embodiments, the present invention
A first Cas9 fusion construct bound to the first half of the inducible heterodimer, and a second Cas9 fusion construct bound to the second half of the inducible heterodimer,
Provided a non-naturally occurring or engineered inducible Cas9 CRISPR-Cas system, including
The first Cas9 fusion construct is operably linked to one or more nuclear localization signals.
The second Cas9 fusion construct is operably linked to the extranuclear translocation signal and
Upon contact with the inducer energy source, the first and second halves of the inducible heterodimer become one.
When the first and second halves of the inducible heterodimer are integrated, the first and second Cas9 fusion constructs can constitute a functional Cas9 CRISPR-Cas system.
The Cas9 CRISPR-Cas system contains a guide RNA (gRNA) containing a guide sequence capable of hybridizing to a target sequence at the target genomic locus of the cell, and the functional Cas9 CRISPR-Cas system edits the genomic locus. Alters gene expression.

In some embodiments, the invention provides a method of treating a subject in need thereof, the method of which is the subject of a polynucleotide as discussed herein or any vector discussed herein. Includes a step of inducing gene editing by transforming and administering an inducer energy source to the subject. The present invention includes the use of such polynucleotides or vectors in the manufacture of a medicament, eg, for treating a subject, or for such a method of treating a subject. The present invention is a method of treating a subject in need thereof, the polynucleotide as discussed herein, or discussed herein, used in a method comprising the step of inducing gene editing. Including any vector, where the method further comprises the step of administering an inducer energy source to the subject. In some embodiments, in this method a repair template is also provided, which is delivered, for example, by a vector containing said repair template.

The present invention also provides a method of treating a subject in need thereof, the method of transforming the subject with the polynucleotides discussed herein or any vector discussed herein. The polynucleotide or vector encodes or encodes a catalytically inactive Cas9 and one or more associated functional domains as discussed herein, comprising the step of inducing transcriptional activation or inhibition by. Included; The method further comprises the step of administering to the subject an inducer energy source. The present invention is also a method of treating a subject in need thereof, the polynucleotide discussed herein or discussed herein, used in a method comprising the step of inducing transcriptional activation or inhibition. Any vector to be provided is also provided, the method further comprising administering to the subject an inducer energy source.

Thus, the present invention specifically has one or more NLS and / or one or more NES, homodimers and heterodimers, dead Cas9 or Cas9 that has essentially no CRISPR activity, eg, by mutation. , System or complex; one or more functional domains linked to split Cas9; including methods and uses, including therapeutic methods.

When referring to Cas9, Cas9 protein or Cas9 enzyme herein, it will be understood that this includes the present split Cas9. In one aspect, the invention provides a method of altering or modifying the expression of a gene product. The method introduces into a cell containing and expressing a DNA molecule encoding a gene product an engineered non-naturally occurring Cas9 CRISPR-Cas system containing a Cas9 protein and a guide RNA that targets the DNA molecule. The guide RNA can target the DNA molecule encoding the gene product, and the Cas9 protein cleaves the DNA molecule encoding the gene product, thereby altering the expression of the gene product; and Here, the Cas9 protein and guide RNA do not coexist in nature. The present invention includes a guide RNA containing a guide sequence linked to a direct repeat (DR) sequence. The present invention further includes Cas9 proteins that are codon-optimized for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

In one aspect, the invention provides an engineered non-naturally occurring Cas9 CRISPR-Cas system comprising a Cas9 protein and a guide RNA that targets a DNA molecule encoding a gene product in the cell, thereby guiding the RNA. However, the DNA molecule encoding the gene product is targeted, and the Cas9 protein cleaves the DNA molecule encoding the gene product, thereby altering the expression of the gene product; and where the Cas9 protein and the guide RNA are Not present together in nature; this includes this split Cas9. The present invention includes a guide RNA containing a guide sequence linked to a DR sequence. The present invention further includes Cas9 proteins that are codon-optimized for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

In another embodiment, the invention is operably linked to a Cas9 protein with a first regulatory element operably linked to a Cas9 CRISPR-Cas system guide RNA that targets a DNA molecule encoding a gene product. Provided is an engineered non-naturally occurring vector system comprising one or more vectors comprising a second regulatory element; this includes the present split Cas9. The components (a) and (b) may be located on the same or different vectors of the system. The guide RNA targets the DNA molecule encoding the gene product in the cell, and the Cas9 protein cleaves the DNA molecule encoding the gene product, thereby altering the expression of the gene product; and where the Cas9 protein And guide RNA do not exist together in nature. The present invention includes a guide RNA containing a guide sequence linked to a DR sequence. The present invention further includes Cas9 proteins that are codon-optimized for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system is (a) a first operably linked to a DR sequence and one or more insertion sites for inserting one or more guide sequences downstream of the DR sequence. Regulatory element [The guide sequence, when expressed, leads to sequence-specific binding of the Cas9 CRISPR-Cas complex to the target sequence in eukaryotic cells, where the Cas9 CRISPR-Cas complex hybridizes to (1) the target sequence. Includes a guide sequence to soy and (2) Cas9 complexed with the DR sequence]; and (b) an operably linked enzyme-encoding sequence that encodes the Cas9 enzyme, including a nuclear localization sequence. Includes two regulatory elements; where components (a) and (b) are located on the same or different vectors of the system; this includes the present split Cas9. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it leads to sequence-specific binding of the Cas9 CRISPR-Cas complex to different target sequences in eukaryotic cells.

In some embodiments, the Cas9 CRISPR-Cas complex is one or more nuclei of sufficient strength to drive the accumulation of a detectable amount of said Cas9 CRISPR-Cas complex in the nucleus of eukaryotic cells. Contains localized sequences. Although not desired to be constrained by theory, Cas9 CRISPR-Cas complex activity in eukaryotes does not require a nuclear localization sequence, but when such a sequence is included, especially of nucleic acid molecules in the nucleus. With respect to targeting, it is believed that the activity of the system is enhanced.

In some embodiments, the Cas9 enzyme is Francisella tularensis 1, Francisella tularensis subsp. Novicida, Prebotera albensis (Prevotella) bacteria Lactera lancera Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp.) BV3L6 , Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens (Eubacterium eligens), Moraxera evigens (Eubacterium eligens), Moraxera libera Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prebotella disiens (Prevoterla disiens), and Porphyromonas from Caeca sian, and Porphyromonas. Yes, it may include a mutant Cas9 derived from these organisms. The enzyme may be Cas9 homolog or ortholog. In some embodiments, Cas9 is codon-optimized for expression in eukaryotic cells. In some embodiments, Cas9 leads to breakage of one or two strands at the location of the target sequence. In a preferred embodiment, the chain break is an attachment end type break with a 5'overhang. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the direct repeat has a minimum length of 16 nt and a single stem loop. In a further embodiment, the direct repeat has a length greater than 16 nt, preferably more than 17 nt, and has two or more stem loops or an optimized secondary structure.

In one aspect, the invention is: (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences downstream of the DR sequence. [When expressed, the guide sequence induces a sequence-specific binding of the Cas9 CRISPR-Cas complex to the target sequence in eukaryotic cells, and the Cas9 CRISPR-Cas complex is (1) a guide sequence that hybridizes to the target sequence. , And (2) containing Cas9 complexed with the DR sequence]; and / or (b) a second operably linked to an enzyme coding sequence encoding the Cas9 enzyme containing a nuclear localization sequence. Provide eukaryotic host cells containing regulatory elements. In some embodiments, the host cell comprises components (a) and (b); which comprises the present split Cas9. In some embodiments, the components (a), components (b), or components (a) and (b) are stably integrated into the genome of the host eukaryotic cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it leads to sequence-specific binding of the Cas9 CRISPR-Cas complex to different target sequences in eukaryotic cells. In some embodiments, Cas9 is codon-optimized for expression in eukaryotic cells. In some embodiments, Cas9 leads to breakage of one or two strands at the location of the target sequence. In a preferred embodiment, the chain break is an attachment end type break with a 5'overhang. In some embodiments, Cas9 lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the direct repeat has a minimum length of 16 nt and a single stem loop. In a further embodiment, the direct repeat has a length greater than 16 nt, preferably more than 17 nt, and has two or more stem loops or an optimized secondary structure. In some embodiments, the invention provides a non-human eukaryote, preferably a multicellular eukaryote, comprising a eukaryotic host cell according to any of the described embodiments. In another aspect, the invention provides a eukaryote comprising a eukaryotic host cell according to any of the described embodiments; preferably a multicellular eukaryote. The organism in some embodiments of these embodiments may be an animal; eg, a mammal. In addition, the organism may be an arthropod such as an insect. The organism may also be a plant. In addition, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more of the constituents described herein. In some embodiments, the kit includes a vector system and instructions for use of the kit. In some embodiments, the vector system is (a) a first operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences downstream of the DR sequence. [The guide sequence, when expressed, leads to sequence-specific binding of the Cas9 CRISPR-Cas complex to the target sequence in eukaryotic cells, and the Cas9 CRISPR-Cas complex hybridizes to (1) the target sequence. Includes a guide sequence and (2) Cas9 complexed with the DR sequence]; and / or (b) operably linked to an enzyme coding sequence encoding the Cas9 enzyme, including a nuclear localization sequence. It includes a second adjusting element, and advantageously this includes the present split Cas9. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it leads to sequence-specific binding of the Cas9 CRISPR-Cas complex to different target sequences in eukaryotic cells. In some embodiments, Cas9 comprises one or more nuclear localization sequences of sufficient intensity to drive the accumulation of a detectable amount of said Cas9 in the nucleus of a eukaryotic cell. In some embodiments, the Cas9 enzyme is Francisella tularensis 1, Francisella tularensis subsp. Novicida, Prebotera albensis (Prevotella) bacteria Lactera lancera Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp.) BV3L6 , Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens (Eubacterium eligens), Moraxera evigens (Eubacterium eligens), Moraxera libera Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prebotella disiens (Prevoterla disiens), and Porphyromonas from Caeca sian, and Porphyromonas. Yes, it may include a mutant Cas9 derived from these organisms. The enzyme may be Cas9 homolog or ortholog. In some embodiments, Cas9 is codon-optimized for expression in eukaryotic cells. In some embodiments, Cas9 leads to breakage of one or two strands at the location of the target sequence. In a preferred embodiment, the chain break is an attachment end type break with a 5'overhang. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the direct repeat has a minimum length of 16 nt and a single stem loop. In a further embodiment, the direct repeat has a length greater than 16 nt, preferably more than 17 nt, and has two or more stem loops or an optimized secondary structure.

In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises attaching the Cas9 CRISPR-Cas complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein Cas9. The CRISPR-Cas complex comprises Cas9 forming a complex with a guide sequence that hybridizes to the target sequence within the target polynucleotide, and the guide sequence is linked to a direct repeat sequence. In some embodiments, the cleavage comprises the step of cleaving one or two strands at the position of the target sequence by the Cas9 enzyme; this comprises the present split Cas9. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises the step of repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair comprises one or more nucleotides of the target polynucleotide. It results in mutations involving insertions, deletions, or substitutions of. In some embodiments, the mutation results in one or more amino acid changes in the protein expressed from the gene containing the target sequence. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, where the one or more vectors are Cas9, and a guide sequence linked to a DR sequence. Drives expression of one or more of them. In some embodiments, the vector is delivered to a subject eukaryotic cell. In some embodiments, the modification occurs in the eukaryotic cell in a cell culture. In some embodiments, the method further comprises the step of isolating the eukaryotic cell from the subject prior to the modification. In some embodiments, the method further comprises the step of returning the eukaryotic cell and / or cells derived thereto to the subject.

In one aspect, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding the Cas9 CRISPR-Cas complex to a polynucleotide, which causes an increase or decrease in the expression of the polynucleotide; where Cas9 CRISPR-Cas The complex is a guide sequence that hybridizes to the target sequence within the polynucleotide and comprises Cas9 that forms a complex with the guide sequence linked to the direct repeat sequence; this includes the present split Cas9. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, where the one or more vectors are Cas9, and a guide sequence linked to a DR sequence. Drives expression of one or more of them.

In one aspect, the invention provides a method of creating a model eukaryotic cell containing a mutant disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method is: (a) the step of introducing one or more vectors into eukaryotic cells [one or more vectors are of Cas9, and a guide sequence linked to a direct repeat sequence. Drive one or more expressions]; and (b) the step of binding the Cas9 CRISPR-Cas complex to a target polynucleotide to result in cleavage of the target polynucleotide within the disease gene [Cas9 CRISPR-Cas complex is , (1) a guide sequence that hybridizes to the target sequence within the target polynucleotide, and (2) Cas9 that is complexed with the DR sequence], thereby containing the mutant disease gene. This includes the present split Cas9. In some embodiments, the cleavage comprises the step of cleaving one or two strands at the location of the target sequence with the Cas9 enzyme. In a preferred embodiment, the chain break is an attachment end type break with a 5'overhang. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises the step of repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair comprises one or more nucleotides of the target polynucleotide. It results in mutations involving insertions, deletions, or substitutions of. In some embodiments, the mutation results in one or more amino acid changes in protein expression from the gene containing the target sequence.

In one aspect, the invention provides a method of developing a biologically active agent that regulates cell signaling events associated with a disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method involves contacting the test compound with a model cell of any one of the embodiments described; and (b) a cell signal associated with the mutation of the disease gene. The biologically active agent is developed that comprises detecting changes in readings that are indicators of decreased or increased transduction events, thereby regulating said cell signaling events associated with said disease gene.

In one aspect, the invention provides a recombinant polynucleotide containing a guide sequence downstream of a direct repeat sequence, where the guide sequence, upon expression, Cas9 CRISPR-to the corresponding target sequence present in the eukaryotic cell. It leads to sequence-specific binding of the Cas complex. In some embodiments, the target sequence is a viral sequence that is present in eukaryotic cells. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

In one aspect, the invention provides a method of selecting one or more cells by introducing one or more mutations into one or more intracellular genes, the method of which is one or more. Steps to introduce one or more vectors into cells [where one or more vectors drive expression of one or more of Cas9, a guide sequence linked to a direct repeat sequence, and an editing template; The editing template in contains one or more mutations that negate Cas9 cleavage]; a step of homologous recombination of the editing template with one or more intracellular target polynucleotides to be selected; Cas9 CRISPR -The step of binding the Cas complex to a target polynucleotide to result in cleavage of the target polynucleotide in the gene [where the Cas9 CRISPR-Cas complex hybridizes to (1) the target sequence within the target polynucleotide. Includes a guide sequence and (2) Cas9 complexed with a direct repeat sequence, where binding of the Cas9 CRISPR-Cas complex to a target polynucleotide induces cell death], thereby one It is possible to select one or more cells into which the above mutations have been introduced; this includes the present split Cas9. In another preferred embodiment of the invention, the cell of choice may be a eukaryotic cell. Aspects of the invention allow the selection of specific cells that do not require a two-step process that may include selectable markers or counterselective systems.

The present specification has the phrase "this includes the present split Cas9" or a similar sentence; and this may be the split Cas9 as Cas9 in the embodiments of the present specification is considered herein. It is to show that.

In some embodiments, the invention presents a first Cas9 fusion construct bound to a first half of an inducible heterodimer and a second Cas9 fusion bound to a second half of an inducible heterodimer. For non-naturally occurring or engineered inducible Cas9 CRISPR-Cas systems, including constructs, the first Cas9 fusion construct is operably linked to one or more nuclear localization signals and the second Cas9 fusion. The construct is operably linked to an extranuclear translocation signal, and upon contact with the inducer energy source, the first and second halves of the inducible heterodimer are united, the first and second half of the inducible heterodimer. When the second halves are united, the first and second Cas9 fusion constructs can form a functional Cas9 CRISPR-Cas system, and the Cas9 CRISPR-Cas system becomes the desired genomic locus of the cell. A guide RNA (gRNA) containing a guide sequence capable of hybridizing to a target sequence is contained, and the functional Cas9 CRISPR-Cas system edits the genomic loci to alter gene expression. In certain embodiments of the invention, the first half of the inducible heterodimer is FKBP12, and the second half of the inducible heterodimer is the FRB. In another embodiment of the invention, the inducer energy source is rapamycin.

The inducer energy source may simply be considered an inducer or a dimerizing agent. For consistency, the term "inducible energy source" is used throughout this specification. The inducer energy source (or inducer) acts to reconstitute Cas9. In some embodiments, the inducer energy source integrates the two parts of Cas9 by the action of the two halves of the inducible dimer. Therefore, the two halves of the inducible dimer are united in the presence of an inducer energy source. The two halves of a dimer do not form (dimerize) a dimer without an inducer energy source.

Therefore, the two halves of the inducible dimer work with the inducer energy source to dimerize the dimer. This in turn reconstructs Cas9 by integrating the first and second parts of Cas9.

Each CRISPR enzyme fusion construct contains one part of split Cas9. These are preferably fused to one of the two halves of the dimer via a linker such as the GlySer linker described herein. The two halves of the dimer may be substantially the same two monomers that together form a homodimer, or they together form a heterodimer. It may be a different monomer. Thus, the two monomers can be thought of as one half of a complete dimer.

Cas9 is a split in the sense that the two parts of the Cas9 enzyme contain substantially functional Cas9. This Cas9 may function as a genome-editing enzyme (when forming a complex with the target DNA and guide), such as nickase or nuclease (which cleaves both strands of DNA), or it is essentially typical. It may be dead Cas9, which is a DNA-binding protein with little or no catalytic activity due to one or more mutations in its catalytic domain.

The two parts of the split Cas9 can be thought of as the N'terminal part and the C'terminal part of the split Cas9. This fusion is typically at the Cas9 split point. In other words, the C'terminal of the N'terminal part of split Cas9 fuses with one of the dimer halves, while the N'terminal of the C'terminal part fuses with the other dimer half. ..

Cas9 does not need to be divided in the sense that a new cut point is created. Split points are typically designed in silico and cloned into the construct. Together, the two parts of the split Cas9, the N'terminal part and the C'terminal part, are preferably at least 70% or more of the wild amino acid (or the nucleotide encoding it), preferably the wild amino acid (or it). To form a complete Cas9 containing at least 80% or more, preferably at least 90% or more, preferably at least 95% or more, and most preferably at least 99% or more of the nucleotides encoding. There may be some trimming, and mutants are assumed. The non-functional domain may be completely removed. The important point is that the two parts can be integrated and that the desired Cas9 function is restored or restored.

The dimer may be a homodimer or a heterodimer.

One or more, preferably two NLS, may be operably linked to the first Cas9 construct. One or more, preferably two NESs, may be operably linked to the first Cas9 construct. The NLS and / or NES may preferably be flanked by a split Cas9 dimer (ie, hemidimer) fusion, i.e., one NLS may be located at the N'terminus of the first Cas9 construct, and One NLS may be at the C'terminus of the first Cas9 construct. Similarly, one NES may be located at the N'terminus of the second Cas9 construct, and one NES may be located at the C'terminus of the second Cas9 construct. When referring to the N'terminus or C'terminus, it will be understood that they correspond to the 5'and 3'terminus of the corresponding nucleotide sequence.

A preferred arrangement is that the first Cas9 construct is located at 5'-NLS- (N'terminal Cas9 part) -linker- (first half of the dimer) -NLS-3'. A preferred arrangement is that the second Cas9 construct is located at 5'-NES- (the second half of the dimer) -linker- (C'terminal Cas9 part) -NES-3'. Preferably there is a suitable promoter upstream of each of these constructs. These two constructs may be delivered separately or together.

In some embodiments, one or all of the NES operably linked to the second Cas9 construct may be replaced with NLS. However, this may typically be undesirable, and in other embodiments, the localization signal operably linked to the second Cas9 construct is one or more NES.

It will also be appreciated that NES may be operably linked to the N'terminal fragment of split Cas9, and NLS may be operably linked to the C'terminal fragment of split Cas9. However, an arrangement in which the NLS is operably linked to the N'terminal fragment of the split Cas9 and the NES is operably linked to the C'terminal fragment of the split Cas9 may be preferred.

The NES functions to localize the second Cas9 fusion construct extranuclearly, at least until an inducer energy source is provided (eg, at least until the inducer is supplied with an energy source to perform its function). .. The presence of the inducer stimulates the dimerization of the two Cas9 fusions in the cytoplasm, and it is thermodynamically valuable for the dimerized first and second Cas9 fusions to be localized in the nucleus. It will be something with. Without being bound by theory, Applicants believe that NES sequesteres the second Cas9 fusion into the cytoplasm (ie, extranuclear). The NLS on the first Cas9 fusion localizes it to the nucleus. In either case, Applicants use NES or NLS to shift the equilibrium in the desired direction (equilibrium for nuclear transport). Dimerization typically occurs extranuclearly (a very small portion can occur intranuclearly), and NLS on the dimerized complex shifts the equilibrium of nuclear transport to nuclear localization. Dimerized and thus reconstituted Cas9 invades the nucleus.

Advantageously, Applicants can undo the functionality of the split Cas9. This concept is proved using transient transfection, and dimerization occurs in the background in the presence of an inducer energy source. No activity is seen with separate fragments of Cas9. Expanding this with stable expression by lentivirus delivery then shows that the split Cas9 approach can be used.

This split Cas9 approach is beneficial because it allows Cas9 activity to be inducible and therefore temporally controllable. In addition, various localized sequences (ie, preferably NES and NLS) can be used to reduce background activity from the self-assembled complex. Tissue-specific promoters, such as those for each of the first and second Cas9 fusion constructs, can also be used for tissue-specific targeting, thus resulting in spatial control. Two different tissue-specific promoters can be used as needed to exert finer control. The same approach may be used for developmental stage-specific promoters, or there may be a mixture of developmental stage-specific and tissue-specific promoters, where one of the first and second Cas9 fusion constructs is tissue-specific. It is under the control of a promoter (ie, operably linked to it or contains it), while the other of the first and second Cas9 fusion constructs is under the control of a developmental stage-specific promoter (ie). That is, it is operably connected to it or includes it).

The inducible Cas9 CRISPR-Cas system comprises one or more nuclear localization sequences (NLS) as described herein, eg, operably linked to a first Cas9 fusion construct. These nuclear localization sequences are ideally strong enough to drive the accumulation of a detectable amount of the first Cas9 fusion construct in the eukaryotic nucleus. Although not desired to be constrained by theory, Cas9 CRISPR-Cas complex activity in eukaryotes does not require a nuclear localization sequence, but when such a sequence is included, especially of nucleic acid molecules in the nucleus. It is believed that the activity of the system is enhanced with respect to targeting and helps the operation of this two-part system.

Similarly, a second Cas9 fusion construct is operably linked to the nuclear translocation sequence (NES). In practice, it can be linked to one or more extranuclear translocation sequences. In other words, the number of extranuclear translocation sequences used with the second Cas9 fusion construct is preferably one, two or three. Two are typically preferred, but one is sufficient and is therefore preferred in some embodiments. Suitable examples of NLS and NES are known in the art. For example, the preferred nuclear translocation signal (NES) is the human protein tyrosine kinase 2. The preferred signal can be species specific.

When the FRB and FKBP systems are used, the FKBP is preferably flanking the nuclear localization sequence (NLS). When the FRB and FKBP systems are used, the preferred arrangement is N'terminal Cas9-FRB-NES: C'terminal Cas9-FKBP-NLS. Thus, the first Cas9 fusion construct may contain a C'terminal Cas9 part, and the second Cas9 fusion construct may contain an N'terminal Cas9 part.

Another aspect beneficial to the present invention is that it can be turned on quickly, i.e. it has a quick response. Although not constrained by theory, Cas9 activity is faster by dimerization of existing (already existing) fusion constructs (by contact with inducer energy sources) than by expression (especially translation) of new fusion constructs. It is thought that it can be induced. Therefore, the first and second Cas9 fusion constructs may be expressed in the target cells in advance, i.e. before Cas9 activity is required. Cas9 activity can then be rapidly regulated by time control and then the addition of an inducer energy source, which can ideally be constrained, for example, by expression of Cas9 delivered by a vector (induction of transcription). Acts faster than by (including) (dimers heterodimers, thereby providing Cas9 activity).

The terms Cas9 or Cas9 enzyme and CRISPR enzyme are used interchangeably herein unless otherwise specified.

Applicants demonstrate that Cas9 can be split into two components, which, when reconstituted, reconstitute a functional nuclease. Applicants use the rapamycin-sensitive dimerization domain to create chemical-induced Cas9 for temporal control of Cas9-mediated genome editing and transcriptional regulation. In other words, Applicants can make Cas9 chemically inducible by splitting it into two fragments, and that Cas9 can be controlled and reassembled using a rapamycin-sensitive dimerization domain. Demonstrate. Applicants show that reassembled Cas9 can be used to mediate genome editing (due to nuclease / nickase activity) and transcriptional regulation (so-called "dead Cas9" as a DNA binding domain).

Therefore, the use of rapamycin sensitive dimerization domains is preferred. Reassembly of Cas9 is preferred. Reassembly can be determined by the restoration of binding activity. If Cas9 is a nickase or induces double-strand breaks, suitable comparison percentages compared to wild type are described herein.

Rapamycin treatment can last for 12 days. The dose can be 200 nM. Administration at this time and / or molarity is an example of a dose suitable for a human fetal kidney 293FT (HEK293FT) cell line, which can also be used for other cell lines. This number can be extrapolated, for example, in mg / kg units for in vivo therapeutic applications. However, it is also envisioned that standard dosages for the administration of rapamycin to a subject are also used herein. "Standard dosage" means a dosage of rapamycin under normal therapeutic use or major application (ie, the dose used when administering rapamycin for the prevention of organ rejection).

Note that the preferred arrangement of Cas9-FRB / FKBP pieces is separate and inactive until FRB and FKBP are dimerized by rapamycin induction to reassemble the functional full-length Cas9 nuclease. Deserves. Therefore, the first Cas9 fusion construct bound to the first half of the inducible heterodimer is delivered separately from the second Cas9 fusion construct bound to the first half of the inducible heterodimer. And / or it is preferred to localize separately.

To isolate the Cas9 (N) -FRB fragment in a cytoplasm that is unlikely to dimerize with the nuclear-localized Cas9 (C) -FKBP fragment, the human protein tyrosine is placed on the Cas9 (N) -FRB. It is preferred to use a single nuclear translocation sequence (NES) from (tyrosin) kinase 2 (Cas9 (N) -FRB-NES). In the presence of rapamycin, Cas9 (N) -FRB-NES dimerizes with Cas9 (C) -FKBP-2xNLS to reconstitute the complete Cas9 protein, which directs the equilibrium of nuclear transport towards nuclear translocation. Shifts and DNA targeting becomes possible.

High dosages of Cas9 can exacerbate the indel frequency in off-target (OT) sequences that show little mismatch to the guide strand. Such sequences are particularly susceptible to non-contiguous mismatches and / or outside the seed region of the guide. Therefore, the use of temporal control of Cas9 activity can reduce dosages in long-term expression experiments and thus result in reduced off-target indel compared to constitutively active Cas9.

Virus delivery is preferred. In particular, lentivirus or AAV delivery vectors are envisioned. Applicants create a split-Cas9 wrench virus construct similar to the wrench CRISPR plasmid. The split pieces must be small enough to meet the AAV size limit of about 4.7 kb.

Applicants demonstrate that stable low copy number expression of split Cas9 can be used to induce substantial indels at loci targeted at off-target sites without significant mutations. Applicants clone Cas9 fragments (two parts based on Split 5 described herein).

For example, in addition to Cas9 (C) -FKBP-2xNLS (dead Cas9 (C) -FKBP-2xNLS-VP64), dead Cas9 containing a VP64 transactivation domain can also be used. These fragments reconstitute a catalytically inactive Cas9-VP64 fusion (dead Cas9-VP64). Transcriptional activation is induced by VP64 in the presence of rapamycin, and dimerization of the Cas9 (C) -FKBP fusion with the Cas9 (N) -FRB fusion is induced. In other words, Applicants test the inducibility of split-dead Cas9-VP64 and reveal that split-dead Cas9-VP64 induces transcriptional activation in the presence of rapamycin. Therefore, one or more functional domains, such as transcriptional activators or repressors or nucleases (such as Fok1), may be associated with this inducible Cas9. The functional domain may be attached to or fused with one part of the split Cas9.

A preferred arrangement is that the first Cas9 construct has a 5'-first localization signal- (N'terminal Cas9 part) -linker- (first half of the dimer) -first localization signal. Arranged at -3'and the second Cas9 construct is 5'-the second localization signal- (the second half of the dimer) -the linker- (the C'terminal Cas9 part) -the second Localization signal-placed in functional domain-3'. Here, the functional domain is placed at the 3'end of the second Cas9 construct. Alternatively, the functional domain may be placed at the 5'end of the first Cas9 construct. One or more functional domains may be used at the 3'end, the 5'end, or both ends. Preferably there is a suitable promoter upstream of each of these constructs. These two constructs may be delivered separately or together. Localization signals may be NLS or NES as long as they do not mix with each other on each construct.

In some embodiments, the present invention provides an inducible Cas9 CRISPR-Cas system in which Cas9 has at least 97% or 100% reduction in nuclease activity when compared to a Cas9 enzyme that does not have at least one mutation.

Therefore, it is also preferable that Cas9 is dead Cas9. Ideally, the split should always be such that one or more catalytic domains are unaffected. Dead Cas9 is intended to result in DNA binding but no cleavage or nickase activity.

In some embodiments, the invention provides an inducible Cas9 CRISPR-Cas system as discussed herein in which one or more functional domains are associated with Cas9. This functional domain may be associated (ie, combined or fused) with one or both parts of the split Cas9. There may be one that meets with each of the two parts of the split Cas9. Thus, they can typically be provided as part of the first and / or second Cas9 fusion construct as a fusion within the construct. Functional domains are typically fused via a linker, such as the GlySer linker, as discussed herein. The one or more functional domains may be transcriptional activation domains or repressor domains. They may be different domains, but it is preferred that all functional domains are either activators or repressors and no mixture of these two is used.

The transcription activation domain may include VP64, p65, MyoD1, HSF1, RTA or SET7 / 9.

In some embodiments, the invention provides an inducible Cas9 CRISPR-Cas system as discussed herein in which one or more functional domains associated with Cas9 are transcriptional repressor domains.

In some embodiments, the invention provides an inducible Cas9 CRISPR-Cas system as discussed herein in which the transcriptional repressor domain is the KRAB domain.

In some embodiments, the invention provides an inducible Cas9 CRISPR-Cas system as discussed herein in which the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or SID4X domain.

In some embodiments, the present invention comprises one or more functional domains associated with an adapter protein that are methylase activity, demethylase activity, transcription activation activity, transcription repressor activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA. Provided is an inducible Cas9 CRISPR-Cas system as discussed herein having one or more activities including cleavage activity, DNA integration activity or nucleic acid binding activity.

Histone modified domains are also preferred in some embodiments. An exemplary histone modification domain is discussed below. Transposase domains, HR (homologous recombination) mechanism domains, recombinase domains, and / or integrase domains are also preferred as the functional domain. In some embodiments, the DNA integration activity comprises an HR mechanism domain, an integrase domain, a recombinase domain and / or a transposase domain.

In some embodiments, the invention provides an inducible Cas9 CRISPR-Cas system as discussed herein in which DNA cleavage activity is due to a nuclease.

In some embodiments, the present invention provides an inducible Cas9 CRISPR-Cas system as discussed herein, in which the nuclease contains a Fok1 nuclease.

The use of such functional domains preferred with this split Cas9 system is also described in Konermann et al. (See also "Genome-scale transcriptional activation with an engineered CRISPR-Cas9 complete" in Nature published 11 Dec 2014).

This system can be used with any guide.

In certain embodiments, modified guides can be used. A guide that embodies the teachings of the above-mentioned Konermann Nature 11 Dec 2014 paper is particularly preferred. These guides are modified to add RNA moieties (aptamers) that bind to proteins. Such one or more parts may replace a part of the guide. RNA can then be recognized using the corresponding RNA-binding protein domains and recruited with a guide to functional domains such as those described herein. This is mainly for use with dead Cas9, leading to transcriptional activation or inhibition or DNA cleavage by nucleases such as Fok1. The use of such guides in combination with dead Cas9 is powerful, especially when Cas9 itself is associated with its own functional domain as discussed herein. This tool is particularly useful when dead Cas9 (with or without its own associated functional domain) is induced to reconstitute according to the present invention, i.e. when it is split Cas9.

Similarly, a guide RNA (gRNA) preferred for use in the present invention can include a guide sequence capable of hybridizing to a target sequence at the genomic locus of interest in the cell, where the gRNA is one or more adapter proteins. It is modified by the insertion of one or more individual RNA sequences that bind to, and the adapter protein is associated with one or more functional domains. Cas9 may contain at least one mutation, so the Cas9 enzyme has less than 5% nuclease activity of the Cas9 enzyme without at least one mutation; and / or at least one or more nuclear localization sequences. Can include. It also naturally comprises one or more guide RNAs (gRNAs) containing a guide sequence capable of hybridizing to a target sequence at the target genomic locus of the cell, and a Cas9 enzyme containing at least one nuclear localization sequence. Non-existent or engineered compositions are also provided, where the Cas9 enzyme contains at least one mutation, so that the Cas9 enzyme has less than 5% nuclease activity of the Cas9 enzyme without at least one mutation. At least one gRNA is modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and the adapter proteins are associated with one or more functional domains.

Preferably, the gRNA has been modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins. Insertion of one or more individual RNA sequences that bind to one or more adapter proteins is preferably an aptamer sequence or two or more aptamer sequences specific for the same or different adapter proteins. .. The adapter protein is preferably MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19. , AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, PRR1. In particular, a cell line that stably expresses split-dead Cas9 may be useful.

Applicants demonstrate that Cas9 can be split into two separate fragments, which, when reconstituted using chemical induction, reconstitute a functional full-length Cas9 nuclease. The split Cas9 architecture can be useful for a variety of applications. For example, split Cas9 can enable a genetic strategy that limits Cas9 activity to a cross-sectional cell population by placing each fragment under a different tissue-specific promoter. In addition, different chemical-induced dimerization domains such as APA and gibberellin can also be used.

The inducer energy source is preferably a chemical substance inducer.

The split position or site is where the first part of the Cas9 enzyme is separated from the second part. In some embodiments, the first part comprises or encodes amino acids 1-X, while the second part comprises or encodes amino acids X + 1-to the end. In this example, the numbering is continuous, but this may not always be necessary, because the amino acid (or the nucleotide encoding it) has sufficient DNA binding activity and, if necessary, DNA nickase or cleavage activity. For example, if at least 40%, 50%, 60%, 70%, 80%, 90% or 95% activity is retained compared to wild-type Cas9, then it can be trimmed from any of the split ends. Is.

The exemplary numbering provided herein can be based on wild-type protein, preferably wild-type FnCas9. However, it is envisioned that wild-type Cas9 mutants, such as FnCas9 protein mutants, can be used. The numbering may also not exactly follow the FnCas9 numbering, for example because some N'or C'terminal truncation or deletion may be used, but this is addressed using standard sequence alignment tools. be able to. Orthologs are also preferred as sequence alignment tools.

Thus, the split position can be selected using, for example, conventional techniques in the art based on crystal data and / or computational structure prediction.

For example, computer analysis of the Cas9 nuclease primary structure reveals three distinct regions (Fig. 1). The first is the C-terminal RuvC-like domain, which is the only functionally characterized domain. The second is the N-terminal α-helix region, and the third is the mixed α and β region located between the RuvC-like domain and the α-helix region. Within the Cas9 primary structure, some small stretches of the unstructured region are expected. Unstructured regions that are exposed to the solvent and are not conserved within different Cas9 orthologs can correspond to the preferred sides for splitting (FIGS. 2 and 3).

The table below provides non-limiting potential split regions within As and LbCas9. The split site within such a region may be convenient.

For Fn, As and Lb Cas9 mutants, which position corresponds to a potential split site should be readily apparent, for example based on sequence alignment. For non-Fn, As and Lb enzymes, the crystal structure of the ortholog can be used if there is a relatively high degree of homology between the ortholog and the intended Cas9, or even with computational prediction. Good.

Ideally, the split position should be located within the region or loop. Preferably, the split position is where disruption of the amino acid sequence does not result in partial or complete disruption of structural features (eg, α-helix or β-sheet). Unstructured regions (regions that do not appear in the crystal structure because they are not sufficiently structured to be "frozen" in the crystal) are often the preferred choice. Applicants may, for example, create splits in unstructured regions exposed on the surface of Cas9.

Applicants may follow the procedure provided as a preferred example and as a guide. Since the unstructured region does not appear in the crystal structure, Applicants cross-reference the amino acid sequences around the crystal with the Cas9 primary amino acid sequence. Each unstructured region can be composed of, for example, about 3-10 amino acids, which do not appear in the crystal. Therefore, Applicants make splits between these amino acids. Applicants include splits located in loops outside Cas9 using the same criteria as for unstructured regions so that more potential split sites are included.

In some embodiments, the split position (positon) is outside the Cas9 loop. In another preferred embodiment, the split position is in the unstructured region of Cas9. The unstructured region is typically an extremely flexible outer loop whose structure cannot be easily determined from the crystal pattern.

Once the split position is identified, a suitable construct can be designed.

Typically, the NES is located at the N'terminus of the first part of the split amino acid (or the 5'terminus of the nucleotide encoding it). In that case, the NLS is located at the C'terminus of the second part of the split amino acid (or the 3'terminus of the nucleotide encoding it). In this way, the first Cas9 fusion construct may be operably linked to one or more extranuclear translocation signals, and the second Cas9 fusion construct may be operably linked to a nuclear localization signal. May be good.

Of course, the reverse configuration may be provided, where the NLS is located at the N'terminus of the first part of the split amino acid (or the 5'terminus of the nucleotide encoding it). In that case, the NES is located at the C'terminus of the second part of the split amino acid (or the 3'terminus of the nucleotide encoding it). Thus, the first Cas9 fusion construct may be operably linked to one or more nuclear localization signals, and the second Cas9 fusion construct may be operably linked to an extranuclear translocation signal. Good.

For packaging, a split that keeps the two parts (both sides of the split) approximately the same length can be advantageous. For example, when transcripts are about the same size, it may be easier to maintain stoichiometry between the two pieces.

In a particular example, codon-optimized human Cas9, such as the N-terminal and C-terminal pieces of FnCas9, fuse into the FRB and FKBP dimerization domains, respectively. This arrangement may be preferred. These may be replaced (ie, FKBP at the N'terminus and FRB at the C'terminus).

In this specification, a linker such as (GGGGS) ₃ is preferably used to separate the Cas9 fragment from the dimerization domain. (GGGGS) ₃ is preferred because it is a relatively long linker (15 amino acids). Glycine residues are the most mobile, and serine residues increase the likelihood that the linker is outside the protein. (GGGGS) ₆ (GGGGS) ₉ or (GGGGS) ₁₂ can preferably be used as an alternative. Other preferred alternatives are (GGGGS) ₁ , (GGGGSS) ₂ , (GGGGGS) ₄ , (GGGGGS) ₅ , (GGGGGS) ₇ , (GGGGSS) ₈ , (GGGGGS) ₁₀ , or (GGGGGS) ₁₁ .

For example, (GGGGS) ₃ may be included between the N'terminal Cas9 fragment and the Fed. For example, (GGGGS) ₃ may be included between the FKB and the C'terminal Cas9 fragment.

Alternative linkers are available, but we believe that highly mobile linkers work best to maximize the chances that the two parts of Cas9 will come together and thus Cas9 activity be restored. Be done. One alternative is that the NLS of nucleoplasmin can be used as a linker.

Linkers can also be used between Cas9 and any functional domain. Again, the (GGGGS) ₃ linker (or its 6, 9, or 12 repeat version) may be used here, or the NLS of nucleoplasmin between Cas9 and the functional domain may be used as the linker. Can be done.

An alternative example of the FRB / FKBP system is assumed. For example, ABA and gibberellin.

Therefore, a preferred example of the FKBP family is any one of the following inducible systems: FKBP dimerizing with calcineurin A (CNA) in the presence of FK506; FKBP dimerizing with CyP-Fas in the presence of FKCsA; FKBP dimerizing with FRB in the presence of rapamycin; coumamycin GyrB dimerizes with GryB in the presence of GyrB; GAI dimerizes with GID1 in the presence of gibererin; or Snap tag dimerizes with HaloTag in the presence of HaXS.

Alternative examples within the FKBP family itself are also preferred. For example, an FKBP that homodimers in the presence of FK1012 (ie, one FKBP dimerizes with another). Therefore,
A first Cas9 fusion construct bound to the first half of the inducible homodimer, and a second Cas9 fusion construct bound to the second half of the inducible homodimer,
Non-naturally occurring or engineered inducible Cas9 CRISPR-Cas systems, including, are also provided.
The first Cas9 fusion construct is operably linked to one or more nuclear localization signals.
The second Cas9 fusion construct is operably linked to (optionally one or more) extranuclear translocation signals.
Upon contact with the inducer energy source, the first and second halves of the inducible homodimer become one.
When the first and second halves of the inducible homodimer are integrated, the first and second Cas9 fusion constructs can form a functional Cas9 CRISPR-Cas system.
The Cas9 CRISPR-Cas system contains a guide RNA (gRNA) containing a guide sequence capable of hybridizing to a target sequence at the target genomic locus of the cell, and the functional Cas9 CRISPR-Cas system binds to the target sequence. And optionally, the genomic loci are edited to alter gene expression.

In one embodiment, the homodimer is preferably FKBP and the inducer energy source is preferably FK1012. In another embodiment, the homodimer is preferably GryB, and the inducer energy source is preferably coumamycin. In another embodiment, the homodimer is preferably ABA and the inducer energy source is preferably gibberellin.

In other embodiments, the dimer is a heterodimer. A preferred example of a heterodimer is one of the following inducible systems: FKBP dimerizing with calcineurin A (CNA) in the presence of FK506; CyP-Fas and dimer in the presence of FKCsA. FKBP to dimerize; FKBP to dimerize with FRB in the presence of rapamycin in the presence of coumamycin; GAI to dimerize with GID1 in the presence of gibererin; or HaloTag and dimer in the presence of HaXS A Snap tag to be quantified.

Applicants used FKBP / FRB because FKBP / FRB is well characterized and both domains are sufficiently small (<100 amino acids) to aid in packaging. In addition, rapamycin has been used for a long time and its side effects are well understood. Large dimerization domains (> 300aa) should also work, but longer linkers may be needed to allow Cas9 reconstruction.

Paulumurugan and Gambir (Cancer Res, August 15, 2005 65; 7413) discusses the background of the FRB / FKBP / rapamycin system. Another useful paper is Crabtree et al. This is an article by (Chemistry & Biology 13, 99-107, Jan 2006).

In one example, a single vector, an expression cassette (plasmid) is constructed. The gRNA is under the control of the U6 promoter. Two different Cas9 splits are used. The split Cas9 construct was a first Cas9 fusion construct in which FKBP was fused to the C-terminal part of the split Cas9 via the GlySer linker; and the N-terminal part of the split Cas9 was fused to the FRB via the GlySer linker. Is based on a second Cas9 fusion construct adjacent to the NES. P2A, which splits during transcription, is used to separate the first and second Cas9 fusion constructs. Split Cas9 exhibits similar indel formation to the wild type in the presence of rapamycin, but in the absence of rapamycin, indel formation is significantly reduced compared to the wild type.

Therefore, a single vector is provided. This vector
A first Cas9 fusion construct bound to the first half of the inducible dimer, and a second Cas9 fusion construct bound to the second half of the inducible dimer,
Including
The first Cas9 fusion construct is operably linked to one or more nuclear localization signals.
The second Cas9 fusion construct is operably linked to one or more extranuclear translocation signals.
Upon contact with the inducer energy source, the first and second halves of the inducible heterodimer become one.
When the first and second halves of the inducible heterodimer are integrated, the first and second Cas9 fusion constructs can constitute a functional Cas9 CRISPR-Cas system.
The Cas9 CRISPR-Cas system contains a guide RNA (gRNA) containing a guide sequence capable of hybridizing to a target sequence at the target genomic locus of the cell, and the functional Cas9 CRISPR-Cas system binds to the target sequence. And optionally, the genomic loci are edited to alter gene expression. These elements are preferably provided in a single construct, eg an expression cassette.

The first Cas9 fusion construct is preferably flanked by at least one nuclear localization signal at each end. The second Cas9 fusion construct is preferably flanked by at least one extranuclear translocation signal at each end.

Also provided is a method of treating a subject in need thereof, which method induces gene editing by transforming the subject with a polynucleotide encoding the system or any of the present vectors, and inducer energy. Includes the step of administering the source to the subject. Suitable repair templates can also be provided, which are delivered, for example, by a vector containing the repair template.

Also provided is a method of treating a subject in need thereof, which is a step of inducing transcriptional activation or inhibition by transforming the subject with a polynucleotide encoding the system or any of the vectors. There is a step in which the polynucleotide or vector encodes or comprises Cas9 and one or more related functional domains that are catalytically inactive; the method administers to an inducer energy source. Includes more steps.

Compositions containing this system used in the therapeutic method are also provided. The use of this system in the manufacture of pharmaceuticals for such therapeutic methods is also provided.

Examples of conditions that can be treated by the system are described herein or in references cited herein.

The single vector can include a transcript splitting agent, such as P2A. P2A divides the transcript in two to separate the first and second Cas9 fusion constructs. The division is due to "ribosome skipping". In essence, the ribosome skips amino acids during translation, which breaks the protein chain and results in two separate polypeptides / proteins. Single vectors are also useful for applications where low background activity is not a concern, but high inducible activity is desired.

One example is the creation of cloned embryonic stem cell lines. The usual procedure is transient transfection with a plasmid encoding wt Cas9 or Cas9 nickase. These plasmids produce Cas9 molecules, which remain active for several days, more likely to be off-target activity. Using a single expression vector for split Cas9 makes it possible to limit "high" Cas9 activity to a shorter time window (eg, one dose of inducer such as rapamycin). Without continuous (daily) induction (eg, rapamycin) treatment, the activity of the single-expressed split Cas9 vector is low, resulting in a reduced likelihood of unwanted off-target effects.

Induced peaks of Cas9 activity are beneficial in some embodiments, which can most easily be achieved using a single delivery vector, but in a dual vector system (each vector is one half of split Cas9). It is also possible by (delivering). The peaks may be highly active and on a short time scale, typically over the life of the inducer.

Therefore, a method for producing a cloned embryonic stem cell line is provided, in which one or more embryonic stem cells are transfected with a polynucleotide encoding the strain or one of the vectors to obtain the split Cas9. It includes a step of expression and a step of inducing Cas9 rearrangement by administering or contacting one or more stem cells with the present inducer energy source. A repair template may be provided.

It will be appreciated that as with any method described herein, a suitable gRNA or guide may be required.

If functional domains or the like are "associating" with either part of the enzyme, they are typically fusions. The term "associating with" is used herein with respect to how one molecule is "associating" with another, for example, between a Cas9 part and a functional domain. In the case of such protein-protein interactions, this association may be considered in terms of recognition in the way antibodies recognize epitopes. Alternatively, one protein may be associated with another protein via a fusion of the two, for example one subunit may be fused to another. Fusion typically occurs by adding one amino acid sequence to the other amino acid sequence, eg, by splicing the nucleotide sequences encoding each protein or subunit together. Alternatively, it may be considered essentially as a bond or direct link between two molecules, such as a fusion protein. In any case, the fusion protein may contain a linker between the two subunits of interest (ie, between the enzyme and the functional domain or between the adapter protein and the functional domain). Thus, in some embodiments, the Cas9 part is associated with it by binding to a functional domain. In other embodiments, Cas9 is associated with the functional domain because the two are optionally fused together via an intermediate linker. Examples of linkers include the GlySer linkers discussed herein.

Other examples of inducers include light and hormones. For light, the inducible dimer may be a heterodimer, the first photoinducible half of the dimer and the second (and complementary) photoinducible half of the dimer. And include. Preferred examples of the first and second photoinduced dimer halves are the CIB1 and CRY2 systems. The CIB1 domain is a heterodimer binding partner for photosensitive cryptochrome 2 (CRY2).

In another example, a blue photoresponsive magnet dimerization system (pMag and nMag) can be fused to two parts of the split Cas9 protein. In response to light stimulation, pMag and nMag are dimerized and Cas9 is reconstituted. For example, for such a system, Nihongaki et al. (Nat. Biotechnology. 33, 755-790, 2015) describes it in relation to Cas9.

The present invention includes that the inducer energy source can be heat, ultrasonic, electromagnetic energy or chemical. In a preferred embodiment of the invention, the inducer energy source may be an antibiotic, small molecule, hormone, hormone derivative, steroid or steroid derivative. In a more preferred embodiment, the inducer energy source may be abscisic acid (ABA), doxycycline (DOX), kumat, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. In the present invention, at least one switch is an antibiotic-based inducible system, an electromagnetic energy-based inducible system, a small molecule-based inducible system, a nuclear receptor-based inducible system, and a hormone-based inducible system. Provided that it can be selected from the group consisting of. In a more preferred embodiment, at least one switch is a tetracycline (Tet) / DOX-inducible system, a photo-inducible system, an ABA-inducible system, a cumatripler / operator system, a 4OHT / estrogen-inducible system, an ecdison-based induction It may be selected from the group consisting of a sex system and an FKBP12 / FRAP (FKBP12-rapamycin complex) inducible system. Such inducers are also discussed herein and in PCT / US2013 / 051418 (incorporated herein by reference).

In general, any possible use of Cas9, whether wt, nickase or dead Cas9 (with or without associated functional domains), can be explored using this split Cas9 approach. Its benefit remains the inducible nature of Cas9 activity.

As a further example, a split Cas9 fusion with a fluorescent protein such as GFP can be made. This allows imaging of genomic loci (“Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR / Cas System” by the optimized CRISPR / Cas system). (see et al. Cell 2013), however, it may be possible in a inducible way. Thus, in some embodiments, one or more of the Cas9 parts can be associated with (and specifically fused with) a fluorescent protein, such as GFP.

Further experiments address whether there is a difference in off-target cutting between wild-type (wt) and split Cas9 when the on-target cutting is at the same level. To do this, Applicants use transient transfection of the wt and split Cas9 plasmids and recover at various time points. Applicants examine off-target activation after finding a set of samples with on-target cleavage within ± 5%. Applicants create cell lines that stably express wt or split Cas9 (using a wrench virus) without guidance. After antibiotic selection, guides are delivered with a separate wrench virus and collected at various time points to measure on / off target amputation.

Applicants have destabilized sequences on the FRB (N) Cas9-NES fragment (PEST, "mRNA destabilizing and protein destabilizing elements for the development of highly responsive reporter systems (Use of mRNA-and protein). -Destabilizing elements to develop a highly respond reporter system) "Von DC et al. Nucleic Acids Research 2005) is introduced to promote the split dead Cas9-V. ..

Such destabilizing sequences (including PEST) as described elsewhere herein may be advantageous for use with the split Cas9 system.

Create a cell line that stably expresses split-dead Cas9-VP64 and MS2-p65-HSF1 + guide. PLX resistance screening can demonstrate that irreversible timed transcriptional activation can be useful in drug screening. This approach can be advantageous when the split dead Cas9-VP64 is not reversible.

In one aspect, the invention provides a non-naturally occurring or engineered Cas9 CRISPR-Cas system that may include at least one switch, wherein the activity of the Cas9 CRISPR-Cas system induces at least one with respect to the switch. It is controlled by contact with a material energy source. In certain embodiments of the invention, control over the activity of at least one switch or said Cas9 CRISPR-Cas system may be activated, enhanced, terminated, or suppressed. Contact with at least one inducer energy source can produce a first effect and a second effect. The first effect is intranuclear translocation, extranuclear translocation, recruitment of secondary components (such as effector molecules), conformational change (of protein, DNA or RNA), cleavage, release of cargo (caged molecule or cofactor). Etc.), may be one or more of associations or dissociations. The second effect may be at least one switch or one or more of the activation, enhancement, termination or suppression of control over the activity of the Cas9 CRISPR-Cas system. In one embodiment, the first and second effects can occur in cascade.

In another aspect of the invention, the Cas9 CRISPR-Cas system is composed of at least one nuclear localization signal (NLS), nuclear localization signal (NES), functional domain, mobile linker, mutation, deletion, It may further include changes or sequences. One or more of the NLS, NES or functional domains may be conditionally activated or inactivated. In another embodiment, the mutation is a mutation in the transcription factor homologous region, a mutation in the DNA binding domain (such as a mutated basic residue in a basic helix loop helix), a mutation in an endogenous NLS or an intrinsic cause. It may be one or more of the mutations in sex NES. The present invention includes that the inducer energy source can be heat, ultrasonic, electromagnetic energy or chemical. In a preferred embodiment of the invention, the inducer energy source may be an antibiotic, small molecule, hormone, hormone derivative, steroid or steroid derivative. In a more preferred embodiment, the inducer energy source may be abscisic acid (ABA), doxycycline (DOX), kumat, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. In the present invention, at least one switch is an antibiotic-based inducible system, an electromagnetic energy-based inducible system, a small molecule-based inducible system, a nuclear receptor-based inducible system, and a hormone-based inducible system. Provided that it can be selected from the group consisting of. In a more preferred embodiment, at least one switch is a tetracycline (Tet) / DOX-inducible system, a photo-inducible system, an ABA-inducible system, a cumatripler / operator system, a 4OHT / estrogen-inducible system, an ecdison-based induction It may be selected from the group consisting of a sex system and an FKBP12 / FRAP (FKBP12-rapamycin complex) inducible system.

A mode of control as detailed in the present application relates to at least one or more switches. The term "switch", as used herein, works cooperatively to influence changes, including all aspects of a biological function, such as activation, suppression, enhancement or termination of that function. Refers to a system or set of components. In one aspect, the term switch comprises a genetic switch comprising the basic constituents of gene regulatory proteins and the specific DNA sequences recognized by these proteins. In one aspect, the switch relates to inducible and inhibitory systems used in gene regulation. In general, an inducible system can be off unless there is some molecule (called an inducer) that allows gene expression. This molecule is said to "induce expression." The method of causing this depends on the control mechanism and the difference in cell type. Inhibitory systems are on except in the presence of some molecule (called a corepressor) that suppresses gene expression. This molecule is said to "suppress expression". The method of causing this depends on the control mechanism and the difference in cell type. The term "inducible", as used herein, may include any aspect of the switch regardless of the molecular mechanism involved. Thus, switches as embraced in the present invention are, but are not limited to, antibiotic-based inducible systems, electromagnetic energy-based inducible systems, small molecule-based inducible systems, and nuclear receptor-based induction. Sexual and hormone-based inducible systems can be included. In a preferred embodiment, the switch is a tetracycline (Tet) / DOX-induced system, a photo-induced system, an abscisic acid (ABA) -induced system, a cumatripler / operator system, a 4OHT / estrogen-induced system, an ecdison-based induction. It may be a sex system or an FKBP12 / FRAP (FKBP12-rapamycin complex) inducible system.

The Cas9 CRISPR-Cas system can be designed to precisely regulate or alter the expression of individual endogenous genes temporally and spatially. The Cas9 CRISPR-Cas system can be designed to bind to the promoter sequence of the gene of interest and alter gene expression. Cas9 may be split in two, where one half fuses with one half of the cryptochrome heterodimer (cryptochrome-2 or CIB1), while the remaining cryptochrome partners are Cas9. Fuse with the other half of. In some embodiments, the transcriptional effector domain may also be included in the Cas9 CRISPR-Cas system. The effector domain can be either an activator such as VP16, VP64, or p65, or a repressor such as KRAB, EnR, or SID. In the unstimulated state, the Cas9-cryptochrome 2 protein in one half is localized to the promoter of the gene of interest but does not bind to the CIB1-effector protein. When stimulated with blue spectral light, cryptochrome-2 is activated to cause a conformational change, exposing its binding domain. Thus, CIB1 binds to cryptochrome-2, resulting in localization of the second half of Cas9 to the promoter region of the gene of interest, initiating genome editing, thereby overexpressing or silencing the gene. Can occur. For aspects of LITE, see Liu, Het al. , Science, 2008 and Kennedy Met al. , Nature Methods 2010, which is incorporated herein by reference in its entirety.

Activator and repressor domains that can further regulate function may be selected based on species, intensity, mechanism, duration, size, or any other parameter. Preferred effector domains are, but are not limited to, transposase domain, integrase domain, recombinase domain, resolverse domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylase. Included are domains, nuclease domains, repressor domains, activator domains, nuclear localization signal domains, transcriptional-protein recruitment domains, cell uptake activity-related domains, nucleic acid binding domains or antibody presentation domains.

In addition, there are several different ways to create chemical-induced systems: 1. 2. ABI-PYL-based system inducible by abscisic acid (ABA) (see, eg, stke.sciencemag.org/cgi/content/abstract/sigtrans; 4/164 / rs2 website). FKBP-FRB-based systems that can be induced by rapamycin (or related chemicals based on rapamycin) (see, eg, nature.com/nmeth/journal/v2/n6/full/nmeth763.html website), 3. 3. A GID1-GAI-based system that can be induced by gibberellin (GA) (see, eg, the website of nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

Another system contemplated by the present invention is a chemical-induced system based on changes in intracellular localization. Applicants also include inducible Cas9 CRISPR-Cas systems engineered to target genomic loci of interest, where Cas9 enzymes are further linked to different parts of the chemical or energy sensitive protein. It is divided into two fusion structures. When a chemical binds to or transfers energy to a chemical or energy-sensitive protein, the chemical or energy-sensitive protein alters the intracellular localization of any half of the Cas9 enzyme (ie, any of the Cas9 enzymes). The half of the body is transported from the cytoplasm of the cell to the nucleus). The fusion construct is thus transported from one intracellular compartment or organelle whose activity is blocked due to the absence of a substrate for the reconstituted Cas9 CRISPR-Cas system to another where the substrate is present. The components can then come together to reconstitute functional activity and then contact the desired substrate (ie, genomic DNA in the mammalian nucleus) to activate or suppress target gene expression. become.

Other inducible systems, but not limited to, regulation by heavy metals [Mayo KE et al. , Cell 1982, 29: 99-108; Searle PF et al. , Mol Cell Biol 1985, 5: 1480-1489 and Brinster RL et al. , Nature (London) 1982, 296: 39-42], Steroid Hormone [Hynes NE et al. , Proc Natl Acad Sci USA 1981, 78: 2038-2042; Klock G et al. , Nature (London) 1987, 329: 734-736 and Lee F et al. , Nature (London) 1981, 294: 228-232. ], Heat shock [Nouer L: Heat Shock Response. Boca Raton, FL: CRC; 1991] and others have been engineered and other reagents have been developed [Mullick A, Massie B: Transcrition, translation and the control of gene expression. In Encyclopedia of Cell Technology Encyclopedia Edited by: Spear RE. Wiley; 2000: 1140-1164 and Fussenegger M .. Biotechnol Prog 2001, 17: 1-51]. However, these inducible mammalian promoters have limitations such as "leakability" in the "off" state and the pleiotropy effect of inducers (heat shock, heavy metals, glucocorticoids, etc.). The use of insect hormones (ecdysone) has been proposed to reduce interference with cellular processes in mammalian cells [No D et al. , Proc Natl Acad Sci USA 1996, 93: 3346-3351]. Another elegant system uses rapamycin as an inducer [Rivera VM et al. , Nat Med 1996, 2: 1028-1032], but because the role of rapamycin as an immunosuppressant was a major constraint on its in vivo use, thus for the regulation of gene expression, biologically inactive compounds. Needed to be found [Saez E et al. , Proc Natl Acad Sci USA 2000, 97: 14512-14517].

See also the following section for inductive systems.

Stabilized Enzymes: Enzymes of the Invention Having or Associated with Destabilizing Domains In one aspect, the invention is non-naturally occurring or engineered with at least one destabilizing domain (DD). CRISPR enzymes, preferably class 2 CRISPR enzymes, preferably V-type or VI-type CRISPR enzymes as described herein, such as preferably, but not limited to, described elsewhere herein. Provided as Cas9; and for simplicity, such non-naturally occurring or engineered CRISPR enzymes associated with at least one destabilizing domain (DD) are referred to herein as "DD-CRISPR enzymes". It is called. Any of the CRISPR enzymes according to the invention as described elsewhere herein may be used as having or associated with a destabilizing domain as described below herein. Should be understood. Any of the methods, products, compositions and uses as described elsewhere herein are equally applicable to CRISPR enzymes associated with destabilizing domains as further detailed below.

As further guidance, the following detailed embodiments and embodiments are provided.

Without the prefix "DD" when aspects and embodiments as described in this section relate to the DD-CRISPR enzyme, DD-CRISPR enzyme, DD-cas9Cas9, DD-CRISPR-Cas or DD-CRISPR-Cas9 system or complex. The terms "CRISPR", "Cas", "Cas9", "CRISPR system", "CRISPR complex", "CRISPR-Cas", "CRISPR-Cas9", etc. are particularly contextual as this disclosure can be read in the embodiments of DD. When recognized above, it can be considered to have the prefix DD. Thus, in one aspect, the invention provides a method of using one or more elements of a CRISPR system, which can be read as a DD-CRISPR system and / or a CRISPR system. The CRISPR complex of the present invention provides an effective means of modifying a target polynucleotide. The CRISPR complex of the present invention has a wide range of usefulness, including modifying (eg, deleting, inserting, translocating, inactivating, activating) target polynucleotides of a wide variety of cell types. Therefore, the CRISPR complex of the present invention has wide applicability in, for example, gene therapy, drug screening, disease diagnosis, and prognosis determination.

In one aspect, the invention is the DD-CRISPR enzyme, eg, a DD-CRISPR enzyme such that the CRISPR enzyme is a Cas protein (referred to herein as "DD-CRISPR enzyme", ie, "DD-CRISPR-". "DD" before terms such as "Cas9 complex" means a CRISPR-Cas9 complex with a Cas9 protein associated with at least one destabilizing domain), advantageously at least one destabilizing domain. Contains a DD-Cas protein associated with, eg, a Cas9 protein (referred to herein as the "DD-Cas9 protein"), and a guide RNA that targets a nucleic acid molecule, such as a DNA molecule, thereby providing a guide RNA. Provided is an engineered non-naturally occurring DD-CRISPR-Cas system that targets a nucleic acid molecule, such as a DNA molecule. Nucleic acid molecules, such as DNA molecules, can encode gene products. In some embodiments, the DD-Cas protein can cleave the DNA molecule encoding the gene product. In some embodiments, the expression of the gene product is altered. Cas protein and guide RNA do not coexist in nature. The present invention includes a guide RNA comprising a guide sequence fused to a tracr sequence where applicable optionally. The present invention further comprises coding codon-optimized Cas proteins for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. Expression of gene products can be reduced. The CRISPR enzyme may form part of the CRISPR-Cas system, which further comprises a guide RNA (gRNA) containing a guide sequence capable of hybridizing to a target sequence at the target genomic locus of the cell. Including. In some embodiments, the functional CRISPR-Cas system binds to the target sequence. In some embodiments, the functional CRISPR-Cas system may edit the target sequence, eg, the target sequence may contain a genomic locus, and in some embodiments there may be changes in gene expression. .. In some embodiments, the functional CRISPR-Cas system may include additional functional domains. In some embodiments, the invention provides a method of altering or modifying the expression of a gene product. The method may include the step of introducing into a target nucleic acid, eg, a cell containing a DNA molecule, or a target nucleic acid, eg, a cell containing and expressing a DNA molecule; eg, the target nucleic acid encodes or genes for a gene product. It can result in expression of the product (eg, regulatory sequence).

In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is a subtype VA or V-B CRISPR enzyme. In some embodiments, the DD-CRISPR enzyme is Cas9. In some embodiments, the DD-CRISPR enzyme is As DD-Cas9. In some embodiments, the CRISPR enzyme is Lb DD-Cas9. In some embodiments, the DD-CRISPR enzyme cleaves both strands of DNA to produce a double-strand break (DSB). In some embodiments, the DD-CRISPR enzyme is nickase. In some embodiments, the DD-CRISPR enzyme is a dual nickase. In some embodiments, the DD-CRISPR enzyme has nuclease activity when compared to dead Cas9, such as Cas9, which has substantially no nuclease activity, such as wild Cas9 or Cas9 which has never had a mutation to it. Is Cas9 of 5% or less. Suitable Cas9 mutations are described elsewhere herein, eg, D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A relative to amino acid positions in the FnCas9p RuvC domain. And N1257A; or, for example, N580A, N584A, T587A, W609A, D610A, K613A, E614A, D616A, K624A, D625A, relative to a presumptive second nuclease domain as described elsewhere herein. Examples include K627A and Y629A.

In some common embodiments, the DD-CRISPR enzyme is associated with one or more functional domains. In some more specific embodiments, the DD-CRISPR enzyme is dead Cas9 and / or is associated with one or more functional domains. In some embodiments, the DD-CRISPR enzyme comprises, for example, an α-helix or α / β mixed secondary structure truncation. In some embodiments, truncation involves removal or replacement with a linker. In some embodiments, the linker is branched or otherwise allows the tethering of DD and / or functional domains. In some embodiments, the CRISPR enzyme is associated with DD via a fusion protein. In some embodiments, the CRISPR enzyme is fused to DD. In other words, DD may be associated with the CRISPR enzyme by fusion with the CRISPR enzyme. In some embodiments, the enzyme may be considered a modified CRISPR enzyme, where the CRISPR enzyme is fused to at least one destabilizing domain (DD). In some embodiments, the DD may associate with the CRISPR enzyme via a connector protein, for example using a system such as a marker system such as a streptavidin-biotin system. Thus, a fusion of a connector protein specific for a high affinity ligand for the connector and a CRISPR enzyme is provided, where DD is bound to the high affinity ligand. For example, streptavidin may be a connector fused to a CRISPR enzyme, while biotin may be bound to DD. When coexisting, streptavidin binds to biotin and thus the CRISPR enzyme binds to DD. For simplicity, fusion of the CRISPR enzyme with DD is preferred in some embodiments. In some embodiments, the fusion comprises a linker between the DD and the CRISPR enzyme. In some embodiments, the CRISPR enzyme may be fused to the N-terminus. In some embodiments, at least one DD fuses to the N-terminus of the CRISPR enzyme. In some embodiments, the CRISPR enzyme may be fused to the C-terminus. In some embodiments, at least one DD fuses to the C-terminus of the CRISPR enzyme. In some embodiments, one DD may be fused to the N-terminus of the CRISPR enzyme and the other DD may be fused to the C-terminus of the CRISPR enzyme. In some embodiments, the CRISPR enzyme associates with at least two DDs, where the first DD fuses to the N-terminus of the CRISPR enzyme and the second DD fuses to the C-terminus of the CRISPR enzyme. The first and second DDs are the same or different. In some embodiments, the DD may be fused to the N-terminus. In some embodiments, the DD may be fused to the C-terminus. In some embodiments, the fusion may be between the C-terminus of the CRISPR enzyme and the N-terminus of the DD. In some embodiments, the fusion may be between the C-terminus of the DD and the N-terminus of the CRISPR enzyme. It was observed that DDs containing at least one N-terminal fusion had a lower background than DDs containing at least one C-terminal fusion. The combination of N-terminal and C-terminal fusion minimized background but had the lowest overall activity. Advantageously, DD is provided by at least one N-terminal fusion or at least one N-terminal fusion + at least one C-terminal fusion. And, of course, DD may be provided by at least one C-terminal fusion.

In certain embodiments, protein destabilizing domains, such as for inducible regulation, can be fused, for example, to the N-terminus and / or C-terminus of Cas9. In addition, the destabilizing domain can be introduced, for example, into the solvent exposure loop of the Cas9 primary sequence. Computer analysis of the primary structure of Cas9 nuclease reveals three distinct regions. The first is the C-terminal RuvC-like domain, which is the only functionally characterized domain. The second is the N-terminal α-helix region, and the third is the mixed α and β region located between the RuvC-like domain and the α-helix region. Within the Cas9 primary structure, some small stretches of the unstructured region are expected. Unstructured regions that are exposed to the solvent and not conserved within different Cas9 orthologs are the preferred sides for splits and insertion of small protein sequences. In addition, these sides can be used to generate chimeric proteins between Cas9 orthologs.

In some embodiments, the DD is ER50. The corresponding stabilizing ligand for this DD is 4HT in some embodiments. Therefore, in some embodiments, one of at least one DD is ER50 and thus the stabilizing ligand is 4HT or CMP8. In some embodiments, the DD is DHFR50. The corresponding stabilizing ligand for this DD is TMP in some embodiments. Therefore, in some embodiments, one of at least one DD is DHFR50 and thus the stabilizing ligand is TMP. In some embodiments, the DD is ER50. The corresponding stabilizing ligand for this DD is CMP8 in some embodiments. Therefore, CMP8 can be an alternative stabilizing ligand for 4HT in the ER50 system. Although it is possible that CMP8 and 4HT can be used / should be used competitively, some cell types may be susceptible to one of these two ligands, and From this disclosure and knowledge in the art, one of ordinary skill in the art can use CMP8 and / or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminus of the CRISPR enzyme and one or two DDs may be fused to the C-terminus of the CRISPR enzyme. In some embodiments, at least two DDs associate with the CRISPR enzyme, and their DDs are the same DD, i.e. the DDs are homologous. Therefore, both (or more than one) DDs can be ER50 DDs. This is preferred in some embodiments. Alternatively, both (or more than one) of the DDs can be DHFR50 DDs. This is also preferable in some embodiments. In some embodiments, at least two DDs associate with the CRISPR enzyme, and their DDs are different DDs, i.e. the DDs are heterologous. Thus, one of the DDs can be the ER50, while one or more of the DDs or any other DD can be the DHFR50. Having two or more DDs that are heterogeneous can be advantageous as they can result in higher degradation control levels. Tandem fusion of two or more DDs at the N-terminus or C-terminus can enhance degradation; and such tandem fusion can be, for example, ER50-ER50-Cas9 or DHFR-DHFR-Cas9. High levels of degradation can occur in the absence of any stabilizing ligand, while intermediate levels of degradation can occur in the absence of one stabilizing ligand and the presence of the other (or another) stabilizing ligand. It is envisioned that low levels of degradation may occur if both (or more than one) of the stabilizing ligands are present. Control can also be provided by having an N-terminal ER50 DD and a C-terminal DHFR50 DD.

In some embodiments, the fusion of CRISPR enzyme and DD comprises a linker between DD and CRISPR enzyme. In some embodiments, the linker is a GlySer linker. In some embodiments, the DD-CRISPR enzyme further comprises at least one nuclear translocation signal (NES). In some embodiments, the DD-CRISPR enzyme comprises more than one NES. In some embodiments, the DD-CRISPR enzyme comprises at least one nuclear localization signal (NLS). This may be included in addition to NES. In some embodiments, the CRISPR enzyme comprises or, as a linker between the CRISPR enzyme and DD, a localization (intranuclear or extranuclear translocation) signal. Or consists of it. HA or Flag tags are also within the scope of the invention as linkers. Applicants use NLS and / or NES as linkers, as well as glycine serine linkers ranging from as short as GS to up to (GGGGS) ₃ .

In some embodiments, the invention provides a polynucleotide encoding a CRISPR enzyme and associated DD. In some embodiments, the encoded CRISPR enzyme and associated DD are operably linked to a first regulatory element. In some embodiments, the DD is similarly coded and operably coupled to a second adjusting element. Advantageously, the DD here is one that "mops up" the stabilizing ligand, and thus advantageously associates with the enzyme, for example as discussed herein. It is understood that the same DD (ie, the same type of domain) (the term "mop up" has the meaning as considered herein and can also convey that an action is taken to contribute to or complete it. Suppose). By mopping up the stabilizing ligand with excess DD that does not associate with the CRISPR enzyme, higher degradation of the CRISPR enzyme will be seen. Although not constrained by theory, the addition of additional or excessive non-associative DD shifts the equilibrium away from the stabilizing ligand that forms a complex with or binds to the DD associated with the CRISPR enzyme, instead. It is envisioned that more will be transferred to the stabilizing ligand that forms or binds to free DD (ie, one that is not associated with the CRISPR enzyme). Therefore, if it is desired to increase the degradation of the CRISPR enzyme but decrease the CRISPR enzyme activity, it is preferable to supply an excess or additional non-associative (or free) DD. Excess free DD binds to the remaining ligands and also takes up ligands bound from the DD-Cas fusion. Therefore, this accelerates DD-Cas degradation and enhances the temporal control of Cas activity. In some embodiments, the first regulatory element is a promoter, which may optionally include enhancers. In some embodiments, the second regulatory element is a promoter and may optionally include enhancers. In some embodiments, the first regulatory element is the initial promoter. In some embodiments, the second regulatory element is the late promoter. In some embodiments, the second regulatory element is an inductive control element, optionally a tet system, or an inhibitory control element, optionally a ttr system, or includes, or essentially from it. Become. An inducible promoter, such as rTTA, may be advantageous for the induction of tet in the presence of doxycycline.

Binding or association can be mediated by a linker, such as a mobile glycine-serine (GlyGlyGlySer) or (GGGS) ₃ , or a strong α-helix linker such as (Ala (GluAlaAlaAlaLys) Ala). In this specification, a linker such as (GGGGS) ₃ is preferably used to separate protein or peptide domains. (GGGGS) ₃ is preferred because it is a relatively long linker (15 amino acids). Glycine residues are the most mobile, and serine residues increase the likelihood that the linker is outside the protein. (GGGGS) ₆ (GGGGS) ₉ or (GGGGS) ₁₂ can preferably be used as an alternative. Other preferred alternatives are (GGGGS) ₁ , (GGGGSS) ₂ , (GGGGGS) ₄ , (GGGGGS) ₅ , (GGGGGS) ₇ , (GGGGSS) ₈ , (GGGGGS) ₁₀ , or (GGGGGS) ₁₁ . Alternative linkers are available, but we believe that highly mobile linkers work best to maximize the chances that the two parts of Cas will come together and thus restore Cas activity. Be done. One alternative is that the NLS of nucleoplasmin can be used as a linker. For example, a linker can also be used between Cas and any functional domain. Again, (GGGGS) ₃ linkers (or 6, 9, or 12 repeat versions thereof) may be used here, or the NLS of nucleoplasmin between Cas and the functional domain may be used as the linker. Can be done.

In some embodiments, the present invention delivers one or more components of the DD-CRISPR-Cas complex of the invention or the polynucleotides discussed herein, eg, one or more components of the complex. Provided are multiple particles, one or more vectors containing one or more polynucleotides discussed herein (eg, encoding a CRISPR enzyme, DD; providing RNA for the CRISPR-Cas complex). To do. In some embodiments, the vector may be a plasmid or viral vector, such as AAV, or a lentivirus. Transient transfection with, for example, HEK cells by plasmid can be reached by limiting the size of AAV, and while Cas9 fits within AAV, with additional coding for association with one or more DDs. Given that, it can be advantageous.

Also provided are models that constitutively express the CRISPR enzyme and associated DD. The organism may be transgenic and may have been transfected with the vector, or may be a progeny of such transfected organism. In a further aspect, the invention provides a composition comprising the CRISPR enzyme described herein and related DDs or polynucleotides or vectors. Also provided is a CRISPR-Cas system containing a guide RNA.

Also provided is a method of treating a subject, eg, a subject in need thereof, which method induces gene editing by transforming the subject with a polynucleotide encoding this system or any of the present vectors. And the step of administering the stabilizing ligand to the subject. Suitable repair templates can also be provided, which are delivered, for example, by a vector containing the repair template. Also provided is a method of treating a subject, eg, a subject in need thereof, which induces transcriptional activation or inhibition by transforming the subject with a polynucleotide encoding the system or any of the vectors. The polynucleotide or vector comprises or comprises a catalytically inactive CRISPR enzyme and one or more related functional domains; the method administers a stabilizing ligand to the subject. Includes more steps. These methods may also include the step of delivering and / or expressing excess DD to the subject. It will be appreciated that the term "subject" can be replaced with the phrase "cell or cell culture" if any treatment is performed in exobibo, eg in cell culture.

Compositions containing this system used in the therapeutic method are also provided. Separate compositions may include stabilizing ligands. A kit of parts containing such a composition may be provided. The use of this system in the manufacture of pharmaceuticals for such therapeutic methods is also provided. The use of this system in screening, such as function acquisition screening, is also provided by the present invention. Cells that are artificially forced to overexpress a gene can down-regulate (rebalance) the gene over time, for example by a negative feedback loop. By the start of screening, unregulated genes can be reduced again. Inducible Cas9 activators allow transcription to be induced just prior to screening, thus minimizing the possibility of false negative hits. Therefore, the possibility of false negative results can be minimized by using the present invention in screening, such as function acquisition screening.

In one aspect, the invention provides an engineered non-naturally occurring CRISPR-Cas system, which comprises a DD-Cas protein and a guide RNA that targets a DNA molecule encoding an intracellular gene product. , Thereby the guide RNA targets the DNA molecule encoding the gene product, and the Cas protein cleaves the DNA molecule encoding the gene product, thereby altering the expression of the gene product; and where the Cas protein And guide RNA do not exist together in nature. The present invention includes a guide RNA containing a guide sequence fused to a tracr sequence. In certain embodiments of the invention, the Cas protein is a Cas9 protein. The present invention further comprises coding codon-optimized Cas proteins for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

If functional domains or the like are "associating" with either part of the enzyme, they are typically fusions. The term "associating with" is used herein with respect to how one molecule is "associating" with another, eg, between a part of a CRISPR enzyme and a functional domain. These two can be considered to be tethered to each other. In the case of such protein-protein interactions, this association may be considered in terms of recognition in the way antibodies recognize epitopes. Alternatively, one protein may be associated with another protein via a fusion of the two, for example one subunit may be fused to another. Fusion typically occurs by adding one amino acid sequence to the other amino acid sequence, eg, by splicing the nucleotide sequences encoding each protein or subunit together. Alternatively, it may be considered essentially as a bond or direct link between two molecules, such as a fusion protein. In any case, the fusion protein may contain a linker between the two subunits of interest (eg, between the enzyme and the functional domain or between the adapter protein and the functional domain). Thus, in some embodiments, the part of the CRISPR enzyme is associated with it by binding to a functional domain. In other embodiments, the CRISPR enzyme is associated with the functional domain because the two are optionally fused together via an intermediate linker. Examples of linkers include the GlySer linkers discussed herein. Non-covalently bound DDs may be able to initiate degradation of associated Cass (eg, Cas9), but proteasome degradation is involved in protein chain unwinding; and DD binds to Cass during degradation. Fusion is preferred as it can provide staying. However, in the presence of a DD-specific stabilizing ligand, the CRISPR enzyme and DD combine to form a stabilizing complex. This complex comprises a stabilizing ligand bound to DD. This complex also includes DD associated with the CRISPR enzyme. In the absence of the stabilizing ligand, degradation of DD and its associated CRISPR enzyme is promoted.

The destabilizing domain has general utility in imparting instability to a wide range of proteins; eg, Miyazaki, JAm Chem Soc. Mar 7, 2012; 134 (9): 3942-3945 (incorporated herein by reference). CMP8 or 4-hydroxytamoxifen can be the destabilizing domain. More generally, temperature-sensitive mutants (DHFRts) of mammalian DHFR, which are destabilizing residues by the N-terminal rule, were found to be stable at acceptable temperatures but unstable at 37 ° C. Addition of methotrexate, a high affinity ligand for mammalian DHFR, to cells expressing DHFRts partially inhibited protein degradation. This was an important demonstration that small molecule ligands can stabilize proteins that are originally targeted for degradation in cells. The use of rapamycin derivatives stabilized the unstable mutant of the FRB domain (FRB *) of mTOR and restored the function of the fused kinase GSK-3β 6,7. This system demonstrated that ligand-gated stability corresponds to an attractive strategy for regulating the function of specific proteins in a complex biological environment. The system that regulates protein activity may involve DD, which becomes functional when ubiquitin complementarity occurs by dimerization of rapamycin-induced FK506-binding protein with FKBP12. Mutants of the human FKBP12 or ecDHFR protein can be engineered to be metabolically unstable in the absence of their high affinity ligands, Shield-1 or trimethoprim (TMP), respectively. These mutants are part of a possible destabilizing domain (DD) useful in the practice of the present invention, and the instability of DD as a fusion with the CRISPR enzyme is the whole proteasome fusion protein. It results in CRISPR proteolysis. Shield-1 and TMP bind to and stabilize DD in a dose-dependent manner. The estrogen receptor ligand binding domain (ERLBD, residues 305-549 of ERS1) can also be engineered as a destabilizing domain. Since the estrogen receptor signaling pathway is involved in various diseases such as breast cancer, this pathway has been extensively studied and numerous estrogen receptor agonists and antagonists have been developed. Therefore, compatible pairs of ERLBD and drugs are known. Some ligands bind to the mutant ERLBD but not to the wild-type ERLBD. By using one of these mutation domains encoding three mutations (L384M, M421G, G521R) 12, ERLBD-derived DD with a ligand that does not disrupt the endogenous estrogen susceptibility network. It is possible to adjust the stability. An additional mutation (Y537S) can be introduced to further destabilize the ERLBD and configure it as a potential DD candidate. This quadruple mutant is an advantageous DD development. This mutant ERLBD can be fused to a CRISPR enzyme, and if a ligand is used to regulate or disrupt its stability, the CRISPR enzyme can have DD. Another DD can be a 12 kDa (107 amino acid) tag based on a mutant FKBP protein stabilized by a Shield1 ligand; see, for example, Nature Methods 5, (2008). For example, DD may be a modified FK506-binding protein 12 (FKBP12) that binds to and is reversibly stabilized by a synthetic biologically inactive small molecule, Shield-1; For example, Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ. "Rapid, reversible, and tunable protein to regulute protein function in living cells synthesis general" Cell. 2006; 126: 995-1004; Banaszynski LA, Cellmyer MA, Contag CH, Wandless TJ, Thorne SH. "Chemical control of protein in living mice". "Chemical control of protein and function in living mice". Nat Med. 2008; 14: 1123-1127; Maynard-Smith LA, Chen LC, Banaszynski LA, Ooi AG, Wandless TJ. "A directed approach for engineering manufacturing conditional protein stability using biological silent silent small molecules". The Journal of Biological Chemistry. 2007; 282: 24866-24872; and Rodriguez, Chem Biol. Mar 23, 2012; 19 (3): 391-398, all of which are incorporated herein by reference, and in the selection of DDs to associate with the CRISPR enzyme in the practice of the invention. Can be used in the implementation of. As you can see, knowledge in the art involves a number of DDs, and DDs can advantageously associate with the CRISPR enzyme with a linker, eg, fuse with it, thereby stabilizing the DD in the presence of a ligand. Can and can destabilize the DD in the absence of it, thereby destabilizing the CRISPR enzyme as a whole, or the DD can be stabilized in the absence of the ligand, and the ligand is present DD can destabilize the DD when it is used; it allows the CRISPR enzyme and thus the CRISPR-Cas complex or system so to speak regulated or controlled on or off regulation, thereby allowing the system to eg in vitro or Provide a means of adjusting or controlling in an in vitro environment. For example, when the protein of interest is expressed as a fusion containing a DD tag, it destabilizes intracellularly and is rapidly degraded, for example by the proteasome. Therefore, the absence of stabilizing ligand leads to degradation of D-associated Cas. When the novel DD is fused to the protein of interest, its instability is imparted to the protein of interest, resulting in rapid degradation of the entire fusion protein. The peak activity of Cas is sometimes beneficial in reducing off-target effects. Therefore, short bursts of high activity are preferred. The present invention is capable of providing such peaks. In a sense, the system is inductive. In another sense, the system is suppressed in the absence of a stabilizing ligand and unsuppressed in the presence of a stabilizing ligand. Other benefits of the invention, without wishing to be bound by any theory, and without making any promises, include:
-Dose adjustable (eg, variable CRISPR-Cas system or complex activity can be achieved as opposed to on or off systems).
• Orthogonality, eg, because only the ligand affects its cognate DD, two or more systems can be manipulated independently and / or the CRISPR enzyme is derived from one or more orthologs. Can be done.
-Can be transported, eg, can function in various cell types or cell lines.
・ High speed.
-There is time controllability.
-Being able to decompose Cas makes it possible to reduce background or off-target Cas or Cas toxicity or excessive Cas accumulation.

The DD may be at the N-terminus and / or C-terminus of the CRISPR enzyme, including the DD on one or more sides of the split (as defined elsewhere herein), eg Cas9 (N). ) -Linker-DD-Linker-Cas9 (C) is also one method of introducing DD. In some embodiments, when using the use of only one terminal association of DD for the CRISPR enzyme, it is preferred to use ER50 as the DD. In some embodiments, when both the N-terminus and the C-terminus are used, the use of either ER50 and / or DHFR50 is preferred. Particularly good results were seen with the N-terminal fusion, which is surprising. Having both an N-terminal fusion and a C-terminal fusion can be synergistic. The size of the destabilizing domain varies, but is typically about 100-300 amino acids in size. The DD is preferably an engineered destabilizing protein domain. DDs and methods of making DDs from, for example, high affinity ligands and their ligand binding domains. The present invention can be considered "orthogonal" as only specific ligands stabilize their respective (cognate) DDs and have no effect on the stability of non-cognate DDs. Commercially available DD systems are CloneTech, ProteoTuner ™ system; the stabilizing ligand is Shield1.

In some embodiments, the stabilizing ligand is a "small molecule". In some embodiments, the stabilizing ligand is cell permeable. It has a high affinity for its corresponding DD. Suitable DD-stabilizing ligand pairs are known in the art. In general, stabilizing ligands are
-Natural processing (eg, proteasome degradation), eg, in vivo;
-Mop-up by: Exobibo / cell culture:
It can be removed by providing a preferred binding partner; or by providing an XS substrate (DD without Cas).

In another embodiment, the invention operably encodes a DD-Cas protein with a first regulatory element operably linked to a CRISPR-Cas-based guide RNA that targets a DNA molecule encoding a gene product. Provided is an engineered non-naturally occurring vector system comprising one or more vectors comprising a linked second regulatory element. The components (a) and (b) may be located on the same or different vectors of the system. The guide RNA targets the DNA molecule that encodes the gene product in the cell, and the DD-Cas protein cleaves the DNA molecule that encodes the gene product (which can cleave one or both strands, or It may have substantially no nuclease activity), thereby altering the expression of the gene product; and where the DD-Cas protein and guide RNA are not naturally present together. In certain embodiments of the invention, the DD-Cas protein is a DD-Cas9 protein. The present invention further comprises coding codon-optimized DD-Cas proteins for expression in eukaryotic cells. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises (a) a direct repeat sequence and one or more guide sequences for inserting one or more guide sequences upstream or downstream (whichever is applicable) of the direct repeat sequence. A first regulatory element operably linked to the insertion site [the guide sequence, when expressed, leads to sequence-specific binding of the DD-CRISPR complex to the target sequence in eukaryotic cells, which causes the CRISPR complex to Contains a DD-CRISPR enzyme complexed with a guide sequence that hybridizes to the target sequence]; and (b) encodes the DD-CRISPR enzyme containing at least one nuclear localization sequence and / or at least one NES. Includes a second regulatory element operably linked to the enzyme coding sequence to be used; where components (a) and (b) are located on the same or different vectors of the system. If applicable, tracr sequences may also be provided. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, each of the two or more guide sequences being true when expressed. It leads to sequence-specific binding of the DD-CRISPR complex to different target sequences in nuclear cells. In some embodiments, the DD-CRISPR complex is one or more nuclear stations of sufficient intensity to drive the accumulation of a detectable amount of the CRISPR complex in or out of the nucleus of a eukaryotic cell. Includes a standing sequence and / or one or more NES. Although not desired to be constrained by theory, nuclear localization sequences and / or NES are not necessarily required for DD-CRISPR complex activity in eukaryotes, but include such sequences, especially intranuclear. It is believed that the activity of the system is enhanced with respect to targeting and / or eukaryotic nucleic acid molecules of. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is a DD-Cas9 enzyme. In some embodiments, the DD-Cas9 enzyme is a bacterium Francisella tularensis 1, a subspecies of the bacterium Francisella tularensis subsp. 1, Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp. ) BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligen (Eubacterium eligen), Eubacteria eligen ), Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevoterella disiens (Prevoterla disiens), or Porphyromonas (Dicadia) It is derived from or can contain further mutations or changes in Cas9) of one of these organisms that have been modified to associate with it, or can be chimeric Cas9. The enzyme may be a DD-Cas9 homolog or ortholog. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides in length. In general, and throughout the specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; free-ended (eg, circular) nucleic acid molecules containing one or more free ends; DNA, Nucleic acid molecules containing RNA, or both; and other types of polynucleotides known in the art. Certain vectors are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where the vector is a virus for packaging into viruses (eg, retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus). The derived DNA or RNA sequence is present. Viral vectors also include polynucleotides carried by the virus for transfection into host cells. A particular vector is capable of self-renewal in the host cell into which it is introduced (eg, a bacterial vector with a bacterial origin of replication and an episomal mammalian vector). Other vectors (eg, non-episome mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. In addition, a particular vector can guide the expression of the gene to which it is operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

The recombinant expression vector can contain a form of nucleic acid of the invention suitable for expression of the nucleic acid in the host cell, that is, one in which the recombinant expression vector is operably linked to the nucleic acid sequence to be expressed. It is meant to include the above regulatory elements, which may be selected based on the host cell used for expression. Within the scope of recombinant expression vectors, "operably linked" allows expression of nucleotide sequences (eg, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell). It is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements.

In some embodiments, the host cell is transiently or non-transiently transfected with one or more of the vectors described herein. In some embodiments, the cell is transfected as it is naturally present in the subject. In some embodiments, the cells to be transfected are harvested from the subject. In some embodiments, the cells are derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MALT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC- 3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI- 231, HB56, TIB55, Jarkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryo fibroblasts, 3T3 Switzerland, 3T3-L1, 132-d5 human fetal fibroblasts; 10. 1 mouse fibroblast, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cell, BEAS-2B, bEnd .. 3, BHK-21, BR293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2 , EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jarkat, JY cells, K562 Cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II , MDCK II, MOR / 0.2R, MONO-MAC6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM- 1, NW-145, OPCN / OPCT cell line, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 Included are cell lines, U373, U87, U937, VCaP, Velo cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines are available from a variety of sources known to those of skill in the art (see, eg, the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, cells transfected with one or more of the vectors described herein are used to establish novel cell lines containing one or more vector-derived sequences. In some embodiments, the components of the CRISPR system as described herein are transiently transfected (eg, by transient transfection of one or more vectors, or transfection with RNA). Using cells that have been affected and modified by the activity of the CRISPR complex, new cell lines are established that include cells that contain the modification but lack any other exogenous sequence. In some embodiments, using cells that have been transiently or non-transiently transfected with one or more of the vectors described herein, or cell lines derived from such cells, 1 One or more test compounds are evaluated.

The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, polyadenylation signals and transcription termination signals such as polyU sequences). .. For such adjustment elements, see, for example, Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that induce constitutive expression of nucleotide sequences in many types of host cells and those that induce expression of nucleotide sequences only in specific host cells (eg, tissue-specific regulatory sequences). Tissue-specific promoters are primarily expressed in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg liver, pancreas), or specific cell types (eg lymphocytes). Can be derived. Regulatory elements may also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, and this expression may or may not be tissue- or cell-type specific. In some embodiments, the vector is one or more pol III promoters (eg, 1, 2, 3, 4, 5, or more pol III promoters) and one or more pol II promoters (eg, 1, 2, 3, 4, 5, or more). One, two, three, four, five or more pol II promoters), one or more pol I promoters (eg, 1, 2, 3, 4, 5, or more pol I promoters), Or includes a combination of these. Examples of the pol III promoter include, but are not limited to, the U6 and H1 promoters. Examples of the pol II promoter include, but are not limited to, the retrovirus Raus sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [eg, with the CMV enhancer). See Boshard et al, Cell, 41: 521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. In addition, the term "adjustment element" includes an enhancer element such as WPRE; CMV enhancer; R-U5'segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8 (1), p. 466-472). , 1988); SV40 enhancer; and intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78 (3), p. 1527-31, 1981). Is also included. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to transform, the desired expression level, and the like. The vector can be introduced into a host cell, whereby the transcript, protein, or peptide encoded by the nucleic acid as described herein is a fusion protein or peptide (eg, clustered regular). Shortly spaced Palindrome repeat (CRISPR) transcripts, proteins, enzymes, mutants thereof, fusion proteins thereof, etc.) can be produced.

Advantageous vectors include lentivirus and adeno-associated virus, and the type of such vector can also be selected to target a particular type of cell.

In one aspect, the invention provides a vector comprising a regulatory element operably linked to an enzyme coding sequence encoding a DD-CRISPR enzyme comprising one or more nuclear localization sequences and / or NES. In some embodiments, the regulatory element drives transcription of the DD-CRISPR enzyme in eukaryotic cells, and the DD-CRISPR enzyme accumulates in a detectable amount in the eukaryotic cell and / or nuclei. Transported outside. In some embodiments, the regulatory element is the polymerase II promoter. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is a DD-Cas9 enzyme. In some embodiments, the DD-Cas9 enzyme is a bacterium Francisella tularensis 1, a subspecies of the bacterium Francisella tularensis subsp. 1, Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp. ) BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligen (Eubacterium eligen), Eubacteria eligen ), Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevoterella disiens (Prevoterla disiens), or Porphyromonas (Dicadia) It can contain further changes or mutations in Cas9, and may be chimeric Cas9, derived from (or modified to associate with it). In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially lacks DNA strand cleavage activity (eg, compared to wild-type enzymes or enzymes that do not have mutations or alterations that reduce nuclease activity. Nuclease activity of 5% or less).

In one aspect, the invention is one or more nuclear localizations of sufficient intensity to drive the accumulation of detectable amounts of said DD-CRISPR enzyme in and / or out of the nucleus of eukaryotic cells. A DD-CRISPR enzyme containing a chemical sequence and / or NES is provided. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is a DD-Cas9 enzyme. In some embodiments, the DD-Cas9 enzyme is a bacterium Francisella tularensis 1, a subspecies of the bacterium Francisella tularensis subsp. 1, Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp. ) BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligen (Eubacterium eligen), Eubacteria eligen ), Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevoterella disiens (Prevoterla disiens), or Porphyromonas (Dicadia) It can contain further changes or mutations in Cas9, and may be chimeric Cas9, derived from (or modified to associate with it). In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially lacks DNA strand cleavage activity (eg, compared to wild-type enzymes or enzymes that do not have mutations or alterations that reduce nuclease activity. Nuclease activity of 5% or less).

In one aspect, the invention comprises (a) a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences upstream or downstream (whichever is applicable) of the direct repeat sequence. The first regulatory element operably linked to [the guide sequence, when expressed, induces sequence-specific binding of the DD-CRISPR complex to the target sequence in eukaryotic cells, and the DD-CRISPR complex Contains a DD-CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence]; and / or (b) encodes the DD-CRISPR enzyme containing at least one nuclear localization sequence and / or NES. Provided is a eukaryotic host cell containing a second regulatory element operably linked to an enzyme coding sequence. In some embodiments, the host cell comprises components (a) and (b). If applicable, tracr sequences may also be provided. In some embodiments, the components (a), components (b), or components (a) and (b) are stably integrated into the genome of the host eukaryotic cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it induces sequence-specific binding of the DD-CRISPR complex to different target sequences in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme is one or more of an intensity sufficient to drive the accumulation of a detectable amount of the CRISPR enzyme in and / or out of the nucleus of a eukaryotic cell. Includes nuclear localization sequence and / or extranuclear translocation sequence or NES. In some embodiments, the DD-CRISPR enzyme is Cas9. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the DD-Cas9 enzyme is a bacterium Francisella tularensis 1, a subspecies of the bacterium Francisella tularensis subsp. 1, Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp. ) BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligen (Eubacterium eligen), Eubacteria eligen ), Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevoterella disiens (Prevoterla disiens), or Porphyromonas (Dicadia) It can contain further changes or mutations in Cas9, and may be chimeric Cas9, derived from (or modified to associate with it). In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially lacks DNA strand cleavage activity (eg, compared to wild-type enzymes or enzymes that do not have mutations or alterations that reduce nuclease activity. Nuclease activity of 5% or less). In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides in length. In some embodiments, the invention provides a non-human eukaryote, preferably a multicellular eukaryote, comprising a eukaryotic host cell according to any of the described embodiments. In another aspect, the invention provides a eukaryote comprising a eukaryotic host cell according to any of the described embodiments; preferably a multicellular eukaryote. The organism in some embodiments of these embodiments may be an animal; eg, a mammal. In addition, the organism may be an arthropod such as an insect. The organism may also be a plant. In addition, the organism may be a fungus.

With respect to the use of the CRISPR-Cas system in general, with reference to the literature including patent applications, patents, and publications cited throughout the disclosure, embodiments of the present invention may be used as within those references. Can be mentioned. One or more CRISPR-Cas systems (eg, single or multiplexed) can be used in conjunction with recent advances in crop genomics. Efficient and cost-effective exploration or editing or manipulation of plant genes or genomes using one or more of these CRISPR-Cas systems-eg, fast exploration and / or of plant genes or genomes. Can be performed for selection and / or exploration and / or comparison and / or manipulation and / or transformation; eg, creating, identifying and developing one or more traits or one or more features , Optimized, or donated to one or more plants, or transformed the plant genome. Thus, there may be new plants with improved plant production, new combinations of traits or characteristics, or new plants with enhanced traits. Such one or more CRISPR-Cas systems can be used with respect to plants in site-specific integration (SDI) or gene editing (GE) or any quasi-reverse breeding (NRB) or reverse breeding (RB) technique. .. Regarding the use of the CRISPR-Cas system in plants, the University of Arizona website "CRISPR-PLANT" (http://www.genome.arizona.edu/crispr/) (sponsored by Penn State and AGI) Be done. Embodiments of the invention can be used for genome editing in plants or where RNAi or similar genome editing techniques have been used so far; for example, Nekrasov, "Easy Plant Genome Editing: CRISPR / Cas Systems. Target genome editing (target genome: targeted genome editing in model and crop plants using the CRISPR / Cas system: CRISPR / Cas system: CRISPR / Cas system: CRISPR / Cas system: CRISPR / Cas system: CRISPR / Cas system: -4811-9-39); Brooks, "Efficient genome editing in the first genome editing CRISPR / Cas9 system using the CRISPR / Cas9 system", CRISPR / Cas9 system, CRISPR / Cas9 system 114.247757; Shan, "Targeted genome modification of CRISPR-Cas system using the CRISPR-Cas system", Nature Biotechnol6, 68 (68) Efficient genome editing in plants using the CRISPR / Cas system (CRISPR / Cas system), Cell Research (2013) 23: 1229-1232. doi: 10.1038 / cr. 2013.114; Online Publication 20 August 2013; Xie, "RNA-guided genome editing in plants using a CRISPR-Cas system", Mol Plant. 2013 Nov; 6 (6): 1975-83. doi: 10.1093 / mp / sst119. Epub 2013 Aug 17; Xu, "Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas," CRISPR-Cas 2014), Zhou et al. , "Use of SNPs for both allergic CRISPR mutations in diplomatic mating of the Kimoto perennial plant Populus reveals 4-kumalic acid: CoA ligase specificity and redundancy (Exploting SNPs for CRISPR mutations in the outcrossing woody perennial Populus revolutions 4-commutation: CoA ligase specialty and CRISPR) ”, New Phytologist (2015) available online; host (2015) (Forum) 1-4 Target DNA degradation using a CRISPR device that stably supports the genome (Mutated DNA degradation using a CRISPR device carry carried in the host genome) ”, NATURE COMMUNICATIONS 6: 6989. nature. com / genomecommunications DOI: 10.1038 / ncomms7989; US Pat. No. 6,603,061-Agrobacterium-Mediated Plant Transformation Method; US Pat. No. 7,868,14. No.-Plant Genome Sequences and Uses Thereof and U.S. Patent Application Publication No. 2009/0100536-Transgenic Plants with Agrobacterium (Transgenic Plants with Agrobacterium) The entire content and disclosure of each of these is incorporated herein by reference in their entirety). In the practice of the present invention, Morrell et al, "Crop genomics: advances and applications", Nat Rev Genet. 2011 Dec 29; 13 (2): 85-96 Content and Disclosure; each of which is incorporated herein by reference, including with respect to how embodiments herein can be used with respect to plants. .. Therefore, references to animal cells herein may also be modified as appropriate and applied to plant cells, unless otherwise apparent.

In one aspect, the invention provides a kit comprising one or more of the constituents described herein. In some embodiments, the kit includes a vector system and instructions for use of the kit. In some embodiments, the vector system is (a) a direct repeat sequence and one or more for inserting one or more guide sequences upstream or downstream (whichever is applicable) of the direct repeat sequence. A first regulatory element operably linked to the insertion site [the guide sequence, when expressed, leads to sequence-specific binding of the CRISPR complex to the target sequence in eukaryotic cells, and the CRISPR complex is the target sequence. Includes a CRISPR enzyme complexed with a guide sequence that hybridizes to]; and / or (b) a second operably linked to an enzyme coding sequence encoding the CRISPR enzyme containing a nuclear localization sequence. Includes adjustment elements. If applicable, tracr sequences may also be provided. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein each of these two or more guide sequences is: When expressed, it leads to sequence-specific binding of the CRISPR complex to different target sequences in eukaryotic cells. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient intensity to drive the accumulation of a detectable amount of the CRISPR enzyme in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is Cas9. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Francisella tularensis 1, Francisella tularensis subsp. Novicida, Prebotera albensis (Prevotella) bacteria Lactera lancera Butyrivibrio-proteobacteria class Chika scan (Butyrivibrio proteoclasticus), Pele Gurini bacteria bacteria (Peregrinibacteria bacterium) GW2011_GWA2_33_10, Parc bacteria bacteria (Parcubacteria bacterium) GW2011_GWC2_44_17, Sumisera species (Smithella sp.) SCADC, Ashida Mino genus species (Acidaminococcus sp.) BV3L6 , Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens (Eubacterium eligens), Moraxera eligens, Moraxera libera Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevoterella disiens (Prevoterella disiens) or at least one Domae diaPorca dia Derived from (modified to associate with it), it can contain further alterations or mutations in Cas9, and may be chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme leads to cleavage of one or two strands at the location of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially lacks DNA strand cleavage activity (eg, compared to wild-type enzymes or enzymes that do not have mutations or alterations that reduce nuclease activity. Nuclease activity of 5% or less). In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides in length.

In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding the DD-CRISPR complex to a target polynucleotide, eg, causing cleavage of the target polynucleotide, thereby modifying the target polynucleotide. The DD-CRISPR complex comprises a DD-CRISPR enzyme that hybridizes with a guide sequence that hybridizes to a target sequence within the target polynucleotide, and the guide sequence is linked to a direct repeat sequence. Where applicable, tracr sequences may also be provided (eg, single guide RNA, sgRNA). In some embodiments, the cleavage comprises cleaving one or two strands at the position of the target sequence with the DD-CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises the step of repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair comprises one or more nucleotides of the target polynucleotide. It results in mutations involving insertions, deletions, or substitutions of. In some embodiments, the mutation results in one or more amino acid changes in the protein expressed from the gene containing the target sequence. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, where the one or more vectors are linked to the DD-CRISPR enzyme and direct repeat sequence. Drives expression of one or more of the guide sequences. If applicable, tracr sequences may also be provided. In some embodiments, the vector is delivered to a subject eukaryotic cell. In some embodiments, the modification occurs in the eukaryotic cell in a cell culture. In some embodiments, the method further comprises the step of isolating the eukaryotic cell from the subject prior to the modification. In some embodiments, the method further comprises the step of returning the eukaryotic cell and / or cells derived thereto to the subject.

In one aspect, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises the step of binding a DD-CRISPR complex to a polynucleotide that causes an increase or decrease in expression of the polynucleotide; where the DD-CRISPR complex is present. Includes a DD-CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence within the polynucleotide, and the guide sequence is linked to a direct repeat sequence. If applicable, tracr sequences may also be provided. In some embodiments, the method further comprises the step of delivering one or more vectors to the eukaryotic cell, where the one or more vectors are linked to the DD-CRISPR enzyme and direct repeat sequence. Drives expression of one or more of the guide sequences. If applicable, tracr sequences may also be provided.

In one aspect, the invention provides a method of creating a model eukaryotic cell containing a mutant disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method is: (a) the step of introducing one or more vectors into eukaryotic cells [one or more vectors are DD-CRISPR enzymes, guide sequences linked to direct repeat sequences. It drives expression of one or more of them (tracr sequences may also be provided where applicable)]; and (b) the DD-CRISPR complex is attached to a target polynucleotide, eg, within the disease gene. The DD-CRISPR complex comprises the step of causing cleavage of the target polynucleotide of the DD-CRISPR complex, which comprises a DD-CRISPR enzyme complexed with a guide sequence that hybridizes to the target sequence within the target polynucleotide. Generate model eukaryotic cells containing mutant disease genes. In some embodiments, the cleavage comprises cleaving one or two strands at the position of the target sequence with the DD-CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises the step of repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair comprises one or more nucleotides of the target polynucleotide. It results in mutations involving insertions, deletions, or substitutions of. In some embodiments, the mutation results in one or more amino acid changes in protein expression from the gene containing the target sequence.

In one aspect, the invention provides a method of developing a biologically active agent that regulates cell signaling events associated with a disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method involves contacting the test compound with a model cell of any one of the described embodiments; and (b) a cell signal associated with the mutation of the disease gene. The biologically active agent is developed that comprises the step of detecting a change in reading that is an indicator of decreased or increased transduction events, thereby regulating said cell signaling events associated with said disease gene.

In one aspect, the invention provides a recombinant polynucleotide containing a guide sequence upstream or downstream of the direct repeat sequence (whichever is applicable), where the guide sequence is present in eukaryotic cells when expressed. Leads to sequence-specific binding of the DD-CRISPR complex to the corresponding target sequence. In some embodiments, the target sequence is a viral sequence that is present in eukaryotic cells. If applicable, tracr sequences may also be provided. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

In one aspect, the invention provides a method of selecting one or more cells by introducing one or more mutations into one or more intracellular genes, the method of which is one or more. Steps to introduce one or more vectors into cells [one or more vectors are DD-CRISPR enzymes, guide sequences linked to direct repeat sequences (where applicable, tracr sequences may also be provided), and edits. Drives expression of one or more of the template; the editorial template contains one or more mutations that abolish DD-CRISPR enzyme cleavage]; one or more intracellular targets to be selected Homologous recombination of a polynucleotide and an editing template; a step of binding the CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide in the gene [DD-CRISPR complex is within the target polynucleotide. Contains the DD-CRISPR enzyme complexed with a guide sequence that mutates to the target sequence, and binding of the DD-CRISPR complex to the target polynucleotide induces cell death], thereby one or more. It allows selection of one or more cells into which the mutation has been introduced. In a preferred embodiment, the DD-CRISPR enzyme is DD-Cas9. In another aspect of the invention, the cell selected may be a eukaryotic cell. Aspects of the invention allow the selection of specific cells that do not require a two-step process that may include selectable markers or counterselective systems. The one or more cells may be prokaryotic or eukaryotic cells.

In a further aspect, the invention is a computer-assisted method for identifying or designing a potential compound to be within or bind to a portion of the DD-CRISPR-Cas9 system or its functionality or vice versa (to the desired compound). Manipulation of the potential DD-CRISPR-Cas9 system or a computer-assisted method for identifying or designing a portion of its functionality to bind) or the potential DD-CRISPR-Cas9 system (eg, DD-CRISPR-Cas9 system). In relation to the possible range of predictions-eg, based on crystal structural data or based on Cas9 ortholog data, or where functional groups such as activators or repressors are added to the DD-CRISPR-Cas9 system. Includes computer-assisted methods for identifying or designing in relation to what is possible, or with respect to Cas9 truncation or with respect to the Nickase design), the methods comprising computer systems such as processors, data storage systems, input devices, and output devices. Using a programmed computer containing, for example, (a) DD-CRISPR-Cas9 crystals in different domains based on differences between Cas9 orthologs, eg, in the DD-CRISPR-Cas9 system binding domain, or in lieu of or in addition to. Data containing three-dimensional coordinates of a subset of atoms from or related to the structure, or with structural information from one or more CRISPR-Cas9 system complexes, optionally with respect to Cas9 or with respect to nickase or functional groups. , A step of inputting to a programmed computer using the input device and thereby creating a dataset; (b) a structure stored in the computer data storage system using the processor, eg, DD- With respect to computer databases of structures of compounds that bind to or presumably bind to the CRISPR-Cas9 system or compounds that are desirable to bind to it, or with respect to DD-Cas9 orthologs (eg, as Cas9 or with respect to different domains or regions between Cas9 orthologs). ) Or DD-CRISPR-Cas9 with respect to crystal structure or nickase or with respect to functional groups; (c) using computer methods to bind one or more structures-eg, to the desired structure. Possible DD-CRISPR-Cas9 structure, specific DD-CR DD-CRISPR-Cas9 that can be manipulated, eg, based on data from other parts of the desired structure, DD-CRISPR-Cas9 crystal structure that can bind to the ISPR-Cas9 structure, and / or data from the DD-CRISPR-Cas9 ortholog. Steps to select from the database where to add or mutate a portion of the system, a truncated Cas9, a novel nickase or a particular functional group, or a functional group to the DD-CRISPR-Cas9 system; (d). Steps to build a model of one or more selected structures using computer methods; and (e) output one or more selected structures to the output device; and optionally select The step of synthesizing one or more of the one or more structures made; and optionally, testing one or more of the synthesized choice structures as or in the DD-CRISPR-Cas9 system. Including steps; or the method comprises at least two atoms of the DD-CRISPR-Cas9 crystal structure, eg, the coordinates of at least two atoms in the material cited herein or the DD-CRISPR-Cas9 crystal (crystal). Data from steps that provide at least subdomain coordinates (“selection coordinates”) of the structure, candidate structures that include binding molecules or, for example, other parts of the DD-CRISPR-Cas9 crystal structure and / or Cas9. Steps that provide the structure of a portion of the DD-CRISPR-Cas9 system that can be manipulated based on data from the ortholog, or the structure of the functional group, and the candidate structure is fitted to the coordinates of choice and thereby combined to the desired structure. Possible DD-CRISPR-Cas9 structure, desired structure capable of binding to a particular DD-CRISPR-Cas9 structure, part of a CRISPR-Cas9 system that can be manipulated, truncated Cas9, novel nickase, or a particular functional group, or Obtaining product data with its output, including positions to add functional groups or mutating the DD-CRISPR-Cas9 system; and optionally, one or more compounds from said product data. Includes steps to synthesize, and optionally, to test the synthesized compound as or in the DD-CRISPR-Cas9 system. The steps to be tested may include analyzing the DD-CRISPR-Cas9 system obtained from one or more of the synthesized selection structures, eg, with respect to binding or with respect to performing the desired function. The output of the method described above includes documents such as data transmission, eg telecommunications, telephone, video conferencing, the media, eg presentations such as computer presentations (eg PowerPoint), the Internet, email, computer program (eg WORD) documents. It may include the transmission of information via communication and the like. Therefore, the present invention is also atomic coordinate data based on the materials cited herein, which defines the three-dimensional structure of DD-CRISPR-Cas9 or at least one subdomain thereof, or the structure of CRISPR-Cas9. It also includes computer-readable media containing factor data that can be derived from the materials cited herein. The computer-readable medium may also contain any data from the methods described above. The present invention further relates to atomic coordinate data based on methods, materials cited herein, data defining a three-dimensional structure of DD-CRISPR-Cas9 or at least one subdomain thereof, or the structure of CRISPR-Cas9. Includes a computer system for generating or implementing rational designs as described above, including factor data, including any of the structural factor data that can be derived from the atomic coordinate data of the materials cited herein. To do. The present invention further comprises a method of conducting business, the three-dimensional structure of a computer system or medium or DD-CRISPR-Cas9 or at least one subdomain thereof, or structural factor data of DD-CRISPR-Cas9. A method comprising providing the user with the structure shown in the atomic coordinate data of the material cited herein and the structural factor data derived from it, or the computer medium herein or the data transmission herein.

A "binding site" or "active site" is a site within a binding cavity or region (such as an atom, a functional group of amino acid residues or multiple such atoms and / or groups) that binds to a compound such as a nucleic acid molecule. Possible, includes, or consists essentially of, a site involved in binding. "Fiting" is the determination of the interaction between one or more atoms of a candidate molecule and at least one atom of the structure of the invention by automatic or semi-automatic means, and to what extent such interaction is stable. It means to calculate what you are doing. Interactions include attractive and repulsive forces brought about by electric charges, three-dimensional considerations, and the like. Various computer-based fitting methods are further described. By "root mean square (or rms) deviation", we mean the square root of the arithmetic mean of the square of the deviation from the mean. "Computer system" means hardware means, software means and data storage means used for analysis of atomic coordinate data. Minimal hardware means for computer-based systems of the present invention typically include a central processing unit (CPU), input means, output means and data storage means. Desirably, a display or monitor is provided to visualize the structural data. The data storage means may be RAM or a means of accessing the computer-readable medium of the present invention. Examples of such systems are computers and tablet devices running Unix, Windows or Apple operating systems. "Computer-readable medium" means any one or more media that can be read and accessed directly or indirectly by a computer, eg, to be a medium suitable for use in the computer systems described above. Such media are not limited, but are limited to magnetic storage media such as floppy disks, hard disk storage media and magnetic tapes; optical storage media such as optical disks or CD-ROMs; electrical storage media such as RAM and ROM; thumb drive devices; cloud Hybrids in these categories such as storage devices and magnetic / optical storage media.

In certain embodiments of the invention, variations in conformation in the crystal structure of the components of the DD-CRISPR-Cas9 system or DD-CRISPR-Cas9 system can be important for the function of the DD-CRISPR-Cas system. It provides important and definitive information about the mobility or migration of protein structural regions relative to RNA or DNA) structural regions. The DD-CRISPR-Cas system herein may be further engineered and optimized using the structural information provided for Cas9 in the materials cited herein, and further estimated from this, other CRISPR enzymes, For example, the structure-functional relationship in a DD-CRISPR enzyme system, such as another V-type or VI-type CRISPR enzyme system (eg, another V-type or VI-type DD-CRISPR enzyme system) can be investigated. The present invention includes an optimized functional DD-CRISPR-Cas enzyme system. In particular, the DD-CRISPR enzyme comprises one or more mutations that convert a functional domain exhibiting the desired function into a DNA binding protein to which it can be recruited, added, inserted or bound. In certain embodiments, the CRISPR enzyme comprises one or more mutations in RuvC1 of the DD-CRISPR enzyme and / or other mutations as discussed herein. In some embodiments, the DD-CRISPR enzyme has one or more mutations in the catalytic domain, where transcription leads the guide sequence to sequence-specific binding of the DD-CRISPR complex to the target sequence. , And here the enzyme further comprises a functional domain (eg, to provide or contribute to a destabilized domain). The structural information provided in the materials cited herein makes it possible to investigate guided interactions between target DNA and CRISPR enzymes (eg Cas9; eg DD-CRISPR enzymes, eg DD-Cas9). Yes, it is possible to engineer or alter the sgRNA structure so that the functionality of the entire DD-CRISPR-Cas system is optimized. For example, the insertion of an adapter protein capable of binding RNA may extend the guide loop without colliding with the Cas9 protein. These adapter proteins can further recruit effector proteins or fusions that contain one or more functional domains. The functional domain may include, be intrinsically, or consist of a transcriptional activation domain, such as VP64. The functional domain can include or can be essentially a transcriptional repressor domain, such as KRAB. In some embodiments, the transcriptional repression domain is a SID, or a concatemer of the SID (eg, SID4X), or comprises, or is essentially derived from it. In some embodiments, the functional domain comprises or becomes essential to an epigenetic modifying domain and provides an epigenetic modifying enzyme. In some embodiments, the functional domain comprises or becomes essential, which may be the P65 activation domain.

Aspects of the invention are a guide RNA (gRNA) containing a guide sequence capable of hybridizing to a target sequence at the target genomic locus of the cell and a DD-CRISPR enzyme that may contain at least one or more nuclear localized sequences. Includes non-naturally occurring or engineered compositions that may include, where the DD-CRISPR enzyme contains one or more mutations, thus this enzyme has changed compared to wild-type enzymes. Or have reduced nuclease activity, where at least one loop of gRNA is modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and one or more adapter proteins. Further recruit the heterogeneous functional domains of. In certain embodiments of the invention, the DD-CRISPR enzyme comprises one or more mutations. In another embodiment, the functional domain comprises or becomes essentially a transcription activation domain, such as VP64. In another embodiment, the functional domain comprises or becomes essentially a transcriptional repressor domain, such as a KRAB domain, a SID domain or a SID4X domain. In embodiments of the invention, does one or more heterologous functional domains include methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity? , Have one or more activities that are essentially derived from them or selected from the group consisting of them. In a further embodiment of the invention, the cell is a eukaryotic cell or a mammalian cell or a human cell. In a further embodiment, the adapter proteins are MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, PRR1 are included, are essentially composed of them, or are selected from the group consisting of them. In another embodiment, at least one loop of gRNA is a tetraloop and / or loop 2. One aspect of the invention includes a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing any of the compositions described herein into the cell.

One aspect of the invention is that the above elements are included in a single composition or in individual compositions. These compositions can advantageously be applied to the host to elicit functional effects at the genomic level.

In general, gRNAs are modified to provide specific binding sites (eg, aptamers) for an adapter protein containing one or more functional domains to bind (eg, via a fusion protein). The modified sgRNA is such that when the gRNA forms a DD-CRISPR complex (ie, the gRNA and the DD-CRISPR enzyme bound to the target), the adapter protein binds and the functional domain on the adapter protein has an effective attribution function. It is modified so that it is positioned in a spatial arrangement that is advantageous for becoming. For example, if the functional domain comprises or becomes essentially a transcriptional activator (eg, VP64 or p65), the transcriptional activator is placed in a spatial arrangement in which it can affect the transcription of the target. .. Similarly, the transcription repressor is positioned to favorably affect the transcription of the target, and the nuclease (eg, Fok1) is positioned to favorably cleave or partially cleave the target.

Those skilled in the art allow the binding of the adapter + functional domain, but modifications to the gRNA that do not allow the adapter + functional domain to be properly positioned (eg, due to steric hindrance within the three-dimensional structure of the CRISPR complex) You will understand that it is an unintended modification. One or more modified gRNAs may be modified in tetraloop, stem-loop 1, stem-loop 2, or stem-loop 3, preferably either tetraloop or stem-loop 2, as described herein. , And most preferably both tetraloop and stemloop 2.

As described herein, functional domains include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity. , And molecular switches (eg, photoinducible), which may be intrinsically derived from them, or may be one or more domains from the group consisting of them. In some cases it is advantageous to further provide at least one NLS and / or NES. In some cases, it is advantageous to position the NLS and / or NES at the N-terminus. When two or more functional domains are included, they may be the same or different.

The gRNA can be designed to contain multiple binding recognition sites (eg, aptamers) that are specific for the same or different adapter proteins. The gRNA can be designed to bind to the promoter region upstream of the -1000 to +1 nucleic acid, preferably -200 nucleic acid, at the transcription initiation site (ie, TSS). Positioning in this way improves the functional domains that affect gene activation (eg, transcriptional activators) or gene inhibition (eg, transcriptional repressors). The modified gRNA is one or more target loci contained in the composition (eg, at least one gRNA, at least two gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 It may be one or more modified gRNAs that target (1 gRNA, at least 50 gRNAs).

Furthermore, the DD-CRISPR enzyme with reduced nuclease activity is at least 70%, at least 80%, at least 90%, at least 95%, at least 97 when the nuclease activity is inactivated (eg, when compared to the wild enzyme). % Or 100% nuclease inactivation; or, in other words, approximately 0% of the nuclease activity of the non-mutated or wild-type Cas9 enzyme or CRISPR enzyme, or the non-mutated or wild-type The DD-Cas9 enzyme or DD-CRISPR enzyme), which is about 3% or about 5% or about 10% or less of the nuclease activity of the Cas9 enzyme or CRISPR enzyme, is the most effective. This is possible by introducing mutations into the RuvC nuclease domain of Cas9 and its orthologs. The inactivated CRISPR enzyme comprises one or more associated functional domains (eg, via a fusion protein), eg, at least one destabilizing domain; or, eg, methylase activity, demethylase activity, transcriptional activation activity, etc. Contains or consists of or consists of transcriptional repressor activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switch (eg, photoinducible). It may have one or more domains from the group, eg, those described herein for a modified gRNA adapter protein. Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. When Fok1 is provided, multiple Fok1 functional domains are provided to realize a functional dimer, and gRNAs are described in Tsai et al. Nature Biotechnology, Vol. It is advantageous to be designed to provide appropriate spacing for functional use (Fok1) as specifically described in 32, Number 6, June 2014). The adapter protein can utilize known linkers to add such functional domains. In some cases, it is advantageous to further provide at least one NLS or NES. In some cases, it is advantageous to position the NLS or NES at the N-terminus. When two or more functional domains are included, they may be the same or different. In general, the location of one or more functional domains on the inactivated DD-CRISPR enzyme allows for the correct spatial placement for the functional domains to influence the target with the functional effect of attribution. For example, if the functional domain is a transcriptional activator (eg, VP64 or p65), the transcriptional activator is placed in a spatial arrangement in which it can affect the transcription of the target. Similarly, the transcription repressor is positioned to favorably affect the transcription of the target, and the nuclease (eg, Fok1) is positioned to favorably cleave or partially cleave the target. This may include positions other than the N-terminus / C-terminus of the DD-CRISPR enzyme.

The adapter protein can be any protein that binds to an aptamer or recognition site introduced into the modified gRNA, which affects the target by attribution function when the gRNA is incorporated into the DD-CRISPR complex. It is possible to properly locate one or more functional domains. As described in detail herein, this can be a coat protein, preferably a bacteriophage coat protein. Functional domains that associate with such adapter proteins (eg, in the form of fusion proteins) include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA. One or more domains from the group consisting of or essentially consisting of or consisting of cleavage activity, nucleic acid binding activity, and molecular switches (eg, photoinducible) can be included. Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. If the functional domain is a transcription activator or transcription repressor, it is advantageous that at least NLS or NES is preferably provided at the N-terminus. When two or more functional domains are included, they may be the same or different. The adapter protein can utilize known linkers to bind such functional domains. Such linkers can be used to associate DD with the CRISPR enzyme, or include DD in the CRISPR enzyme.

Thus, gRNAs such as modified gRNAs, inactivated DD-CRISPR enzymes (with or without functional domains), and binding proteins with one or more functional domains are each individually included in the composition and individually. It can be administered to the host either in or in bulk. Alternatively, these components may be provided in a single composition for administration to a host. Administration to the host can be carried out via viral vectors known to those of skill in the art for delivery to the host or described herein (eg, lentivirus vector, adenovirus vector, AAV vector). As described herein, the use of different selectable markers (eg, for lentivirus sgRNA selection) and gRNA concentrations (eg, depending on whether multiple gRNAs are used) can improve efficacy. It can be advantageous to produce. Based on this concept, several variations are appropriate for inducing genomic locus events, including DNA cleavage, gene activation, or gene deactivation. One of ordinary skill in the art will use the provided composition to advantageously and specifically target single or multiple loci having the same or different functional domains to induce one or more genomic locus events. be able to. The composition comprises screening in a library of cells and in vivo functional modeling (eg, gene activation and functional identification of lincRNA; functional acquisition modeling; loss of function modeling; cell lines and transgenic animals for optimization and screening purposes. It can be applied to a wide variety of methods for the use of the compositions of the present invention to establish.

The present invention includes the use of the compositions of the present invention for establishing and utilizing conditioned or inducible CRISPR transgenic cells / animals; eg, Platt et al. , Cell (2014), 159 (2): 440-455, or PCT patent gazettes cited herein, such as WO 2014/093622 (PCT / US2013 / 074667). .. For example, cells or animals such as non-human animals such as vertebrates or mammals such as rodents such as rodents such as mice, rats, or other laboratory or field animals such as cats, dogs, sheep and the like are "knocked in". The animal may express DD-Cas9 conditionally or inducibly as well as Platt et al. Thus, the target cell or animal contains the DD-CRISPR enzyme (eg, DD-Cas9) conditionally or inducibly (eg, in the form of a Cre-dependent construct) and / or conditionally or induces the adapter protein or DD. Upon expression of a vector contained in sex and introduced into a target cell, the vector induces or results in the expression of DD-CRISPR enzyme (eg, DD-Cas9) and / or adapter or DD in the target cell. Expressed as. Inducible genomic events are also aspects of the invention by applying the teachings and compositions of the invention along with known methods of creating CRISPR complexes. A mere example of this is the production of a CRISPR knock-in / conditional transgenic animal (eg, a mouse containing, for example, a Lux-termination-poly A-Lox (LSL) cassette), followed by one or more modified gRNAs (eg, a gene). From -200 nucleotides of the target gene of interest for activation to TSS, eg, a modified gRNA with one or more aptamers recognized by a coat protein, eg MS2), one as described herein. One or more compositions that provide the above adapter proteins (MS2-binding proteins linked to one or more VP64s) and means for inducing conditional animals (eg, Cre recombinases for inducing DD-Cas9 expression). Delivery of goods. Alternatively, the adapter protein or DD may be provided as a conditional or inducible element with a conditional or inducible CRISPR enzyme to provide an effective model for screening purposes, which is advantageous in a wide variety. It requires minimal design and administration of specific gRNAs for various applications.

In some embodiments, especially when a hereditary disease is targeted in a therapeutic method, the phenotypic change is preferably the result of genomic modification, preferably to modify or alter the phenotype here. A repair template is provided.

In some embodiments, potentially targeted diseases include those associated with disease-causing splice deficiency.

In some embodiments, cell targets include hematopoietic stem / progenitor cells (CD34 +); human T cells; and eyes (retinal cells) -eg, photoreceptive progenitor cells.

In some embodiments, the gene target is human β-globin-HBB, including for the treatment of sickle cell anemia, by stimulation of gene conversion (using a closely related HBD gene as an endogenous template); CD3 (T cells); and CEP920-retina (eye).

In some embodiments, disease targets also include cancers that cause splice deficiency; sickle cell anemia (based on point mutations); HBV, HIV; β-thalassemia; and ophthalmic or eye diseases-eg, label congenital. Amaurosis (LCA)-also includes. In some embodiments, delivery methods include cationic lipid-mediated "direct" delivery of enzyme-guide complexes (ribonuclear proteins) and electroporation of plasmid DNA.

The methods, products and uses described herein can be used for non-therapeutic purposes. In addition, any of the methods described herein can be applied in vitro and in exobibo.

In some embodiments
I. Two or more CRISPR-Cas-based polynucleotide sequences
(A) A first guide sequence capable of hybridizing to a first target sequence at a polynucleotide locus,
(B) A second guide sequence capable of hybridizing to a second target sequence at the polynucleotide locus,
(C) Direct repeat array,
(D) A polynucleotide sequence, optionally, including a tracr sequence; and II. Cas9 enzyme or a second polynucleotide sequence encoding it,
Non-naturally occurring or engineered compositions comprising
The Cas9 enzyme is a modifying enzyme that contains one or more DDs as described herein.
When transcribed, the first and second guide sequences lead to sequence-specific binding of the first and second CRISPR complexes to the first and second target sequences, respectively.
The first CRISPR complex comprises a Cas9 enzyme complexed with a first guide sequence capable of hybridizing to a first target sequence.
The second CRISPR complex contains a Cas9 enzyme that has formed a complex with a second guide sequence that is hybridizable to the second target sequence.
The first guide sequence leads to the cleavage of one strand of the DNA duplex near the first target sequence, and the second guide sequence leads to the cleavage of the other strand near the second target sequence. Dechaining is induced, thereby altering an organism or non-human or non-animal organism.

In another embodiment Cas9 is delivered to cells as a protein. In another particularly preferred embodiment, Cas9 is delivered to cells as a protein or as a nucleotide sequence encoding it. Delivery to cells as a protein may include delivery of a ribonucleoprotein (RNP) complex, where the protein forms a complex with a guide.

In certain embodiments, host cells and cell lines that are modified by or contain the compositions, systems or modifying enzymes of the invention, including stem cells and their progeny, are provided.

In some embodiments, a method of cell therapy is provided, wherein, for example, a single cell or cell population is sampled or cultured, wherein the one or more cells are exobibo as described herein. It has been modified or modified and then reintroduced (sampled cells) or introduced (cultured cells) into the organism. Stem cells, whether embryonic stem cells or induced pluripotent or totipotent stem cells, are particularly preferred in this regard. But, of course, in vivo embodiments are also envisioned.

The methods of the invention can further include delivery of a template, such as a repair template, which may be dsODN or ssODN (see below). Delivery of the template may be simultaneous or separate with delivery of some or all of the CRISPR enzyme or guide, and may be by the same or different delivery mechanisms. In some embodiments, the template is preferably delivered with the guide, preferably with the CRISPR enzyme. One example may be an AAV vector, where the CRISPR enzyme is AsCas9 or LbCas9.

The method of the present invention is (a) a step of delivering a double-stranded oligodeoxynucleotide (dsODN) containing an overhang complementary to the overhang caused by the double-strand break to cells, wherein the dsODN is an object. Incorporating into the locus of the gene; or-(b) delivering a single-stranded oligodeoxynucleotide (ssODN) to cells, wherein the ssODN acts as a template for homology-dependent repair of the double-stranded break. Can be further included. The method of the present invention may be a method of preventing or treating a disease of an individual, and the disease is optionally caused by a defect in the locus of interest. The method of the present invention can be performed in vivo in an individual or ex vivo on cells taken from an individual, and the cells are optionally returned to the individual.

The present invention also includes products obtained using the CRISPR enzyme or Cas enzyme or Cas9 enzyme or CRISPR-CRISPR enzyme or CRISPR-Cas system or CRISPR-Cas9 system of the present invention.

Structural Homology; Homologs and Orthologs In embodiments, Cas9 proteins as referred to herein also include Cas9 homologs or orthologs, such as SpCas9 or eSpCas9. The terms "orthologue" (also referred to herein as "ortholog") and "homology" (also referred to herein as "homology") are the techniques of the art. Well known in the field. As a further guide, a protein "homolog" as used herein is a protein of the same species that performs the same or similar function as the protein for which it is a homolog. Homologs and orthologs can be identified by homology modeling (eg, Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or "Structural BLAST (Structural BLAST)". (Day F, Cliff Zhang Q, Petery D, Honig B. "Towards" Structural BLAST ": Inferring Function Using Structural Relationships (Toward a" Structural BLAST ": using scientific relationship" science .Protein Sci. 2013 Apr; 22 (4): 359-66. Doi: 10.10012 / pro.2225). For application in the field of the CRISPR-Cas locus, see Shmakov et al. See also (2015). However, homolog proteins do not have to be structurally related, or are only partially structurally related. A protein "ortholog" as used herein is a different species of protein that performs the same or similar function as the protein for which it is an ortholog. However, ortholog proteins do not have to be structurally related, or are only partially structurally related. In a detailed embodiment, Cas9 homologs or orthologs as referred to herein are sequences such as at least 80%, more preferably at least 85%, even more preferably at least 90%, eg, at least 95% of Cas9. Have homology or identity. In a further embodiment, the Cas9 homolog or ortholog as referred to herein is at least 80%, more preferably at least 85%, even more preferably at least 90%, eg at least 95%, etc. of the wild-type Cas9. Has sequence identity of. In a detailed embodiment, Cas9 homologs or orthologs as referred to herein are sequences such as at least 80%, more preferably at least 85%, even more preferably at least 90%, eg, at least 95% of Cas9. Have homology or identity. In a further embodiment, the Cas9 homolog or ortholog as referred to herein is at least 80%, more preferably at least 85%, even more preferably at least 90%, eg at least 95%, etc. of the wild-type SpCas9. Has sequence identity of. If Cas9 has (mutates) one or more mutations, the homolog or ortholog of said Cas9 as referred to herein is at least 80%, more preferably at least 85, of the mutant Cas9. %, More preferably at least 90%, for example at least 95%.

Specific domains of ortholog proteins are involved as well. In certain embodiments, the ortholog domain of Cas9 as referred to herein has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence homology or identity with Cas9. Has. In a detailed embodiment, the Cas9 ortholog domain as referred to herein has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence homology or identity with SpCas9. Has.

Delivery of CRISPR-Cas9 Complex or Constituents Based on this disclosure and knowledge in the art, the CRISPR-Cas system, especially the novel CRISPR-Cas systems described herein, or their components or nucleic acid molecules thereof (eg HDR templates). Nucleic acid molecules encoding or providing (including) or components thereof can be delivered by the delivery system described herein in general and in detail.

Vector delivery, eg, plasmid, viral delivery: CRISPR enzymes such as Cas9, Cas9 orthologs or variants thereof, and / or any present RNA, eg, a guide RNA, can be any suitable vector, eg, a plasmid or viral vector. , For example, adeno-associated virus (AAV), lentivirus, adenovirus, or other types of viral vectors, or combinations thereof. Cas9 and one or more guide RNAs can be packaged in one or more vectors, such as plasmids or viral vectors. In some embodiments, the vector, eg, a plasmid or viral vector, is delivered to the tissue of interest, eg, by intramuscular injection, while delivery is intravenous, transdermal, intranasal, oral, mucosal, or other. It may depend on the delivery method. Such delivery may be by a single dose or by multiple doses. Those skilled in the art will appreciate that the actual dosage delivered herein is vector selection, target cells, organism or tissue, general condition of the subject to be treated, degree of transformation / modification required, route of administration, mode of administration. Understand that it can vary significantly depending on various factors such as the type of transformation / modification required.

Such doses may include, for example, carriers (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluents, pharmaceutically acceptable carriers (such as sesame oil). For example, phosphate buffered saline), pharmaceutically acceptable excipients, and / or other compounds known in the art may be further included. Dosages include one or more pharmaceutically acceptable salts, such as mineral salts such as hydrochlorides, hydrobromates, phosphates, sulfates; and acetates, propionates, malonates. , Organic salts such as benzoate may be further included. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gels or gelling substances, fragrances, colorants, microspheres, polymers, suspending agents may also be present therein. In addition, one or more other conventional pharmaceutical ingredients such as preservatives, wetting agents, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stability. Agents and the like may also be present, especially if the dosage form is a reconstituted form. Suitable exemplary components include microcrystalline cellulose, sodium carboxymethyl cellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethylvanillin, glycerin, phenol, Examples include parachlorophenol, gelatin, albumin, and combinations thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mac Pub. Co., N.J. 1991) (incorporated herein by reference). ..

In certain embodiments herein, delivery via a adenovirus, which may be a single booster dose containing adenoviral vector of at least 1 × 10 ⁵ particles (grain units, also referred pu). In certain embodiments herein, the dose is preferably at least about 1 × 10 ⁶ particles (eg, about 1 × 10 ⁶ to 1 × 10 ¹² particles), more preferably at least about 1 × 10 ⁷ particles, more preferably. at least about 1 × 10 ⁸ particles (e.g., about 1 × ¹⁰ 8 to 1 × ^{10 11} particles, or about 1 × ¹⁰ 8 to 1 × ^{10 12} particles), and most preferably at least about 1 × 10 ⁰ particle (for example, About 1 x 10 ⁹ to 1 x 10 ¹⁰ particles or about 1 x 10 ⁹ to 1 x 10 ¹² particles), or even at least about 1 x 10 ¹⁰ particles (eg, about 1 x 10 ¹⁰ to 1 x 10 ¹² particles) Adenovirus vector. Alternatively, the dose is about 1 × 10 ¹⁴ particles or less, preferably about 1 × 10 ¹³ particles or less, even more preferably about 1 × 10 ¹² particles or less, even more preferably about 1 × 10 ¹¹ particles or less, and most preferably. Includes about 1 × 10 ¹⁰ particles or less (eg, about 1 × 10 ⁹ particles (articles) or less). Thus, the doses are, for example, about 1 × 10 ⁶ particle units (pu), about 2 × 10 ⁶ pu, about 4 × 10 ⁶ pu, about 1 × 10 ⁷ pu, about 2 × 10 ⁷ pu, about 4 × 10 ⁷ pu, about 1 x 10 ⁸ pu, about 2 x 10 ⁸ pu, about 4 x 10 ⁸ pu, about 1 x 10 ⁹ pu, about 2 x 10 ⁹ pu, about 4 x 10 ⁹ pu, about 1 x 10 ¹⁰ pu, about 2 × 10 ¹⁰ pu, about 4 × 10 ¹⁰ pu, about 1 × 10 ¹¹ pu, about 2 × 10 ¹¹ pu, about 4 × 10 ¹¹ pu, about 1 × 10 ¹² pu, about 2 × 10 ¹² pu Or may contain a single dose of adenovirus vector containing about 4 × 10 ¹² pu of adenovirus vector. For example, Nabel, et., Granted on June 4, 2013. al. U.S. Pat. No. 8,454,972, B2 (incorporated herein by reference), adenovirus vector, and dosages in column 29, lines 36-58. In certain embodiments herein, the adenovirus is delivered in multiple doses.

In certain embodiments herein, delivery is via AAV. Therapeutically effective dosages for in vivo delivery of AAV to humans range from about 20 to about 50 ml saline containing about 1 x 10 ¹⁰ to about 1 x 10 ¹⁰ functional AAV / ml solutions. it is conceivable that. The dosage can be adjusted to balance the therapeutic benefits with any side effects. In certain embodiments herein, AAV doses are generally about 1 × 10 ⁵ to 1 × 10 ⁵⁰ genomic AAV, about 1 × 10 ⁸ to 1 × 10 ²⁰ genomic AAV, about 1 × 10 ¹⁰ to about 1 ×. The concentration range is 10 ¹⁶ genomes, or about 1 × 10 ¹¹ to about 1 × 10 ¹⁶ genomes AAV. The human dosage may be about 1 × 10 ¹³ genomic AAV. Such concentrations can be delivered in about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art using routine testing to create a dose-response curve. See, for example, paragraphs 27-60 of US Pat. No. 8,404,658 B2, granted to Hajjar et al. On March 26, 2013.

In one embodiment of the specification, delivery is via a plasmid. For such plasmid compositions, the dose should be sufficient for the plasmid to elicit a response. For example, a suitable amount of plasmid DNA in a plasmid composition can be about 0.1 to about 2 mg, or about 1 μg to about 10 μg for a 70 kg person. The plasmids of the invention generally include (i) a promoter; (ii) a sequence operably linked to the promoter, encoding a CRISPR enzyme; (iii) a selectable marker; (iv) an origin of replication; and (v) ( Includes a transcription terminator operably linked to (ii) downstream of ii). The plasmid may also encode the RNA component of the CRISPR complex, but one or more of these may be encoded in different vectors instead.

The doses herein are based on an average of 70 kg. The frequency of administration is within the range of medical practitioners or veterinary practitioners (eg, physicians, veterinarians), or those skilled in the art. It should also be noted that the mouse used in the experiment typically weighs about 20 g and can be scaled up to a 70 kg person from the mouse experiment.

In some embodiments, the RNA molecules of the invention are delivered in liposomes, lipofection formulations, etc., and can be prepared by methods well known to those of skill in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference. It is in. Delivery systems have been developed specifically for the delivery of improved and improved siRNAs to mammalian cells (eg, Shen et al FEBS Let. 2003, 539: 111-114; Xia et al., Nat. Biotech. 2002, 20: 1006-1010; Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis et al., Nat Gen. 2002, 32: 107-108, and Simeoni et al., NAR 2003, 31, 11: 2717-2724), which can be applied to the present invention. SiRNA has been successfully used in the suppression of gene expression in primates in recent years (see, for example, Tolentino et al., Retina 24 (4): 660, which can also be applied to the present invention).

In fact, RNA delivery is a useful method of in vivo delivery. Cas9 and gRNA (and, for example, HR repair templates) can be delivered intracellularly using liposomes or particles or nanoparticles. Thus, delivery of CRISPR enzymes, such as Cas9 and / or RNA of the invention, can be carried out in RNA form by microvesicles, liposomes, or particles or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged in liposome particles for in vivo delivery. Liposomal transfection reagents, such as Life Technologies lipofectamines and other reagents on the market, can effectively deliver RNA molecules to the liver.

The means of delivery of RNA is also preferably delivery by particles or nanoparticles of RNA (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev). , S., Ranger, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endoselial cells, Advanced Funcational Cell, Advanced Func. , C., Cortez, C., Ranger, R., and Anderson, D., Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267: 9-21,20 including 267: 9-21,20. In fact, exosomes should be particularly useful for the delivery of systems that have some similarity to siRNA, CRISPR systems. For example, El-Andaloussi S, et al. ("Exosome-mediated delivery of siRNA in vitro and in vivo." Nat Protocol. 2012 Dec; 7 (12): 2112-26. Doi: 10.1038 / nprot. 2012.131. Epub 2012 No. It describes how exosomes are a promising tool for drug delivery across various biological barriers and can be used for in vitro and in vivo delivery of siRNA. In this approach, target exosomes are generated by transfection of an expression vector containing an exosome protein fused to a peptide ligand. The exosomes are then purified and characterized from the transfected cell supernatant, and then RNA is introduced into the exosomes. In particular, but not limited to, delivery or administration according to the present invention to the brain can be performed using exosomes. Vitamin E (α-tocopherol) is conjugated to CRISPR Cas to deliver short interfering RNA (siRNA) to the brain, for example as performed by Uno et al. (HUMAN GENE THERAPY 22: 711-719 (June 2011)). It can be delivered to the brain along with high density lipoprotein (HDL) in a manner similar to that. Infused into mice by osmotic minipump (Model 1007D; Alzet, Cupertino, CA) filled with phosphate buffered saline (PBS) or free TocsiBACE or Toc-siBACE / HDL and connected to Brain Infusion Kit 3 (Alzet). did. A cerebral infusion cannula was placed approximately 0.5 mm after the bregma at the midline for injection into the dorsal third ventricle. Uno et al. Found that only 3 nmol Toc-siRNA containing HDL could result in comparable target reduction by the same ICV injection method. Similar doses of α-tocopherol-conjugated CRISPR Cas co-administered with HDL to target the brain can be contemplated in humans in the present invention, eg, about 3 nmol to about 3 μmol of CRISPR that targets the brain. Cas can be intended. Zou et al. ((HUMAN GENE THERAPY 22: 465-475 (April 2011)) described a method for lentivirus-mediated delivery of PKCγ-targeted short-chain hairpin RNA for gene silencing in vivo in the spinal cord of rats. Described. Zou et al. Administered approximately 10 μl of recombinant lentivirus with a titer of 1 × 10 ⁹ transfection units (TU) / ml via a submembrane catheter. Lentivirus vector targeting the brain. in a similar amount of CRISPR Cas being expressed, resulting contemplated in humans in the present invention, for example, in the lentivirus with a titer of 1 × 10 ⁹ transducing units (TU) / ml, and the target brain About 10 to 50 ml of CRISPR Cas can be contemplated.

This can be achieved in a variety of ways with respect to local delivery to the brain. For example, the substance can be delivered into the striatum, for example by injection. The injection can be performed stereotactically by craniotomy.

Increasing NHEJ efficiency or HR efficiency also helps with delivery. NHEJ efficiency is preferably enhanced by co-expression of terminal processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188 (4): 787-797). HR efficiency is preferably increased by transient inhibition of NHEJ devices such as Ku70 and Ku86. HR efficiency can also be increased by co-expression of prokaryotic or eukaryotic homologous recombinases such as RecBCD, RecA.

Packaging and Promoters How to package a Cas9-encoding nucleic acid molecule of the invention, eg, DNA, into a vector, eg, a viral vector, to mediate genomic modification in vivo:
-Achieve NHEJ-mediated gene knockout:
-Single viral vector:
A vector containing two or more expression cassettes:
-Promoter-Cas9 coding nucleic acid molecule-Terminator:
-Promoter-gRNA1-Terminator:
-Promoter-gRNA2-Terminator:
-Promoter-gRNA (N) -Terminator: (up to vector size limit):
・ Double viral vector:
Vector 1: containing one expression cassette that drives Cas9 expression
-Promoter-Cas9 coding nucleic acid molecule-Terminator:
Vector 2 containing one or more expression cassettes that drive the expression of one or more guide RNAs:
-Promoter-gRNA1-Terminator:
-Promoter-gRNA (N) -Terminator: (up to vector size limit):
• Mediates homology-dependent repair:
• In addition to the single and dual viral vector approaches described above, additional vectors can be used to deliver homology-dependent repair templates.

Promoters used to drive the expression of Cas9-encoding nucleic acid molecules can include: AAV ITR can serve as promoters: it does not require additional promoter elements (which can take up space in the vector). Is advantageous. The additional free space can be used to drive the expression of additional elements (such as gRNA). Also, since ITR activity is relatively weak, it can be used to reduce the potential toxicity due to overexpression of Cas9. For ubiquitous expression, promoters can be used that include:
CMV, CAG, CBh, PGK, SV40, and Ferritin heavy or light chains and the like.

For expression in the brain or other CNS, the following promoters can be used: Synapsin I for any neuron, CaMKII alpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
For expression in the liver, the albumin promoter can be used.
For expression in the lung, SP-B can be used.
For endothelial cells, ICAM can be used.
For hematopoietic cells, IFNβ or CD45 can be used.
In the case of osteoblasts, OG-2 can be used.

Promoters used to drive guide RNA can include:
Pol III promoter, eg U6 or H1
Use of the Pol II promoter and an intron cassette that expresses gRNA.

Adeno-associated virus (AAV)
Cas9 or Cas9 mutants or orthologs and one or more guide RNAs can be used with adeno-associated virus (AAV), lentivirus, adenovirus, or other types of plasmids or viral vectors, in particular, eg, US Pat. 8,454,972 (formulations and doses for adenovirus), 8,404,658 (formulations and doses for AAV), and 5,846,946 (DNA). It can be delivered using formulations and doses from (plasmid formulations, doses) and from publications on clinical trials and clinical trials on lentivirus, AAV, and adenovirus. For example, in the case of AAV, the route of administration, formulation, and dose can be similar to US Pat. No. 8,454,972 and clinical trials for AAV. For adenovirus, the route of administration, formulation, and dose can be similar to US Pat. No. 8,404,658 and clinical trials for adenovirus. For plasmid delivery, the route of administration, formulation, and dose can be similar to US Pat. No. 5,846,946 and clinical studies on plasmids. Doses can be based on or extrapolated on average 70 kg of humans (eg, human adult males) and can be tailored to various body weights and species of patients, subjects, mammals. The frequency of dosing is within the realm of the practitioner or veterinary practitioner (eg, physician, veterinarian), age, gender, general health, other conditions of the patient or subject, and specific conditions to be addressed. Or it depends on normal factors including symptoms. The viral vector can be injected into the tissue of interest. In the case of cell-type-specific genomic modification, Cas9 expression can be driven by a cell-type-specific promoter. For example, albumin promoters can be used for liver-specific expression, and the Synapsin I promoter can be used for neuron-specific expression (eg, when targeting CNS disorders). With respect to in vivo delivery, AAV is more than other viral vectors due to a few reasons: low toxicity, which is obtained by purification methods that do not require ultracentrifugation of cell particles that can activate the immune response. It is advantageous.

Since AAV does not integrate into the host genome, it is unlikely to cause insertion mutations.

AAV has a packaging limit of 4.5 Kb or 4.75 Kb. This means that Cas9 and the promoter and transcription terminator must all fit within the same viral vector. Constructs larger than 4.5 Kb or 4.75 Kb significantly reduce virus production. SpCas9 is fairly large, with the gene itself exceeding 4.1 Kb, making it difficult to pack into AAV. Therefore, embodiments of the present invention include utilizing a relatively short Cas9 homolog. For example:

Therefore, these species are generally preferred Cas9 species.

For AAV, AAV can be AAV1, AAV2, AAV5, or any combination thereof. AAV can be selected for AAV on cells to be targeted; for example, when targeting brain or nerve cells, AAV serotypes 1, 2, 5, or hybrid capsids AAV1, AAV2, AAV5, or these. Any combination of can be selected; AAV4 can be selected when targeting heart tissue. AAV8 is useful for delivery to the liver. The promoters and vectors herein are individually preferred. A table of specific AAV serotypes for these cells (see Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) is as follows:

Lentivirus Lentivirus is a complex retrovirus that has the ability to infect both mitotic and mitotic cells and express its genes. The best-known lentivirus is the human immunodeficiency virus (HIV), which uses enveloped glycoproteins from other viruses to target a wide range of cell types.

Wrench virus would be preferred as follows: After cloning of pCasES10 (including the backbone of the lentivirus transfer plasmid), low passage (p = 5) HEK293FT the day before transfection in antibiotic-free DMEM supplemented with 10% fetal bovine serum. Was seeded in a T-75 flask to 50% confluence. After 20 hours, the medium was replaced with OptiMEM (serum-free) medium and transfection was performed after 4 hours. 10 μg lentivirus transfer plasmid (pCasES10) and the following packaging plasmid: 5 μg pMD 2. Transfected with G (VSV-g pseudoform) and 7.5 μg psPAX2 (gag / pol / rev / tat). Transfection was performed in 4 mL of OptiMEM containing a cationic lipid delivery agent (50 uL of lipofectamine 2000 and 100 ul of Plus reagent). After 6 hours, the medium was replaced with antibiotic-free DMED containing 10% fetal bovine serum. These methods use serum during cell culture, but serum-free methods are preferred.

The lentivirus can be purified as follows. After 48 hours, the virus supernatant was collected. Debris was first removed from the supernatant and filtered through a 0.45 μm low protein binding (PVDF) filter. The supernatant was then ultracentrifuged at 24,000 rpm for 2 hours. Virus pellets were resuspended in 50 μl DMEM overnight at 4 ° C. Then, it was divided into equal parts and quickly frozen at −80 ° C.

In another embodiment, minimal non-primate based on equine infectious anemia virus (EIAV), especially for gene therapy of the eye (see, eg, Balagaan, J Gene Med 2006; 8: 275-285). Similar wrench virus vectors are also contemplated. In another embodiment, it expresses RetinoStat®, ie, the anti-angiogenic proteins endostatin and angiostatin, delivered by subretinal injection for the treatment of reticular (webform) age-related macular degeneration. A lentivirus gene therapy vector based on the horse-infectious anemia virus is also contemplated (eg, Binley et al., HUMAN GENE THERAPY 23: 980-991 (September 2012)), and this vector is the CRISPR-Cas of the present invention. Can be modified for the system.

In another embodiment, a self-inactivating lentiviral vector containing a common exon-targeting siRNA shared by HIV tar / rev, nuclear-localized TAR decoys, and anti-CCR5-specific hammerhead ribozymes (eg,). , DiGiusto et al. (2010) Sci Transl Med 2: 36ra43) can be used and / or adapted to the CCRISP-Cas system of the present invention. Collect at least 2.5 x 106 CD34 + cells per kg body weight of the patient, 2 μmol / L-glutamine, stem cell factor (100 ng / ml), Flt-3 ligand (Flt-3L) (100 ng / ml), and It can be pre-stimulated in X-VIVO15 medium (Lonza) containing thrombopoetin (10 ng / ml) (CellGenix) at a concentration of 2 x 106 cells / ml for 16-20 hours. Pre-stimulated cells can be transduced in a fibronectin-coated 75 cm2 tissue culture flask (25 mg / cm2) (RetroNectin, Takara Bio Inc.) for 16-24 hours with a wrench virus in a quintuple infection. ..

Lentiviral vectors are disclosed in the treatment of Parkinson's disease, see, for example, U.S. Patent Application Publication No. 20120295960 as well as U.S. Pat. Nos. 7,303,910 and 7,351,585. I want to. Wrench viral vectors are also disclosed in the treatment of eye disorders, such as, for example, US Patent Application Publication Nos. 20060281180, 20090007284, 20110117189; US 20090017543; Please refer to the specification of No. 20070054961 and the specification of No. 2010317109. The lentivirus vector is also disclosed for delivery to the brain, eg, US Patent Application Publication No. 201101293571; No. 201101293571, No. 2004013648, No. 20070025970, No. 200901111106. Please refer to the specification and US Pat. No. 7,259,015.

Delivery of RNA Delivery of RNA: CRISPR enzymes, such as Cas9 and / or any of this RNA, such as guide RNA, can also be delivered in the form of RNA. Cas9 mRNA can be made using in vitro transcription. For example, Cas9 mRNA can be synthesized using a PCR cassette containing the T7_promoter-kozak sequence (GCCACC) -Cas9-3'UTR from the following element: β-globin-poly A tail (a series of 120 or more adenines). it can. This cassette can be used for transcription by T7 polymerase. Guide RNA can also be transcribed using in vitro transcription from a cassette containing the T7_promoter-GG-guide RNA sequence.

To promote expression and reduce possible toxicity, the CRISPR enzyme coding sequence and / or guide RNA should include one or more modified nucleotides, eg, with pseudoU or 5-methyl-C. It can be modified.

mRNA delivery methods are currently particularly promising for delivery to the liver.

Although many clinical studies on RNA delivery have focused on RNAi or antisense, these systems can be applied to RNA delivery for the practice of the present invention. The following references, such as RNAi, should be read carefully. In fact, RNA delivery is a useful method of in vivo delivery. Cas9 and gRNA (and eg, HR repair templates) can be delivered intracellularly using liposomes or nanoparticles. Thus, delivery of a CRISPR enzyme, such as Cas9, and / or delivery of the RNA of the invention can be in RNA form via microvesicles, liposomes, or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged in liposome particles for in vivo delivery. Liposomal transfection reagents, such as Life Technologies lipofectamines and other reagents on the market, can effectively deliver RNA molecules to the liver.

As a preferred means of delivering RNA as well as particle delivery of RNA, delivery by nanoparticle of RNA (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y , Bogatirev, S., Langer, R. and Anderson, D., "Lipid-like nanoparticles for small interfering RNA delibery energy for delivery of small interfering RNA to endothelial cells." Functional Materials, 19: 3112-3118, 2010) or delivery by exosomes (Schroeder, A., Levins, C., Cortez, C., Ranger, R., and Anderson, D., "lipid-based for siRNA delivery. Nanotherapy (Lipid-based nanotherapetics for siRNA delivery) ”, Journal of International Medicine, 267: 9-21, 2010, PMID: 20059441) can also be mentioned. In fact, exosomes have been shown to be particularly useful for the delivery of siRNA, a system that has some similarity to the CRISPR system. For example, El-Andaloussi S, et al. ("Exosome-mediated delivery of siRNA in vitro and in vivo" Nat Protoc. 2012 Dec; 7 (12): 2112-26. Doi: 10.1038 / npro. 131. Epub 2012 Nov 15) describes how exosomes are promising tools for drug delivery across various biological barriers and are available for in vitro and in vivo delivery of siRNA. Their approach involves transfection of an expression vector to create a target exosome containing an exosome protein fused to a peptide ligand. The exosomes are then purified from the transfected cell supernatant and characterized, after which RNA is loaded into the exosomes. Delivery or administration according to the present invention can be carried out using exosomes, in detail, but not limited to, to the brain. Vitamin E (α-tocopherol) was conjugated to CRISPR Cas, eg, Uno et al. It can be delivered to the brain along with high density lipoprotein (HDL), similar to the delivery of small interfering RNA (siRNA) to the brain performed by (HUMAN GENE THERAPY 22: 711-719 (June 2011)). .. Mice infused with osmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled with phosphate buffered saline (PBS) or free TocsiBACE or Toc-siBACE / HDL and connected to Brain Infusion Kit 3 (Alzet). Was done. A brain-injection cannula was placed approximately 0.5 mm posterior to the bregma on the midline for injection into the dorsal third ventricle. Uno et al. Found that only 3 nmol of Toc-siRNA containing HDL was able to induce a similar reduction in targets by the same ICV injection method. Similar dosages of CRISPR Cas conjugated to α-tocopherol, co-administered with HDL targeting the brain, can be contemplated in humans in the present invention, eg, about 3 nmol to about 3 μmol targeting the brain. CRISPR Cas can be intended. Zou et al. ((HUMAN GENE THERAPY 22: 465-475 (April 2011)) describes a method for lentivirus-mediated delivery of short-chain hairpin RNA targeting PKCγ for in vivo gene silencing in rat spinal cord. Zou et al. Administered approximately 10 μl of recombinant lentivirus with a titer of 1 × 10 ⁹ silencing units (TU) / ml via a submembrane catheter. Expressed in a brain-targeted lentivirus vector. Similar dosages of CRISPR Cas can be contemplated in humans in the present invention, eg, about 10-50 ml targeting the brain in a lentivirus having a titer of 1 × 10 ⁹ transfection units (TU) / ml. CRISPR Cas can be intended.

With respect to local delivery to the brain, this can be achieved in a variety of ways. For example, the substance can be delivered into the striatum, for example by injection. The injection can be performed stereotactically by craniotomy.

Enhancing NHEJ or HR efficiency also aids in delivery. NHEJ efficiency is preferably enhanced by co-expression of terminal processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188 (4): 787-797). The HR efficiency is preferably increased by transiently inhibiting NHEJ mechanisms such as Ku70 and Ku86. HR efficiency can also be increased by co-expression of prokaryotic or eukaryotic homologous recombinases such as RecBCD, RecA.

Plasmid Delivery In certain embodiments herein, delivery is via a plasmid. In such plasmid compositions, the dosage should be sufficient amount of plasmid to elicit the reaction. For example, a suitable amount of plasmid DNA in a plasmid composition can be from about 0.1 to about 2 mg, or from about 1 μg to about 10 μg, per 70 kg of an individual. The plasmids of the invention generally include (i) a promoter; (ii) a sequence operably linked to the promoter, encoding a CRISPR enzyme; (iii) a selectable marker; (iv) an origin of replication; and (v). It may include a transfer terminator operably linked to (ii) downstream of (ii). The plasmid can also encode the RNA component of the CRISPR complex, but one or more of these may instead be encoded on different vectors.

The doses herein are based on an average of 70 kg individuals. The frequency of administration is within the discretion of a medical or veterinary practitioner (eg, physician, veterinarian) or scientist in the art. It is also noted that the mice used in the experiments typically weigh about 20 g and can be scaled up to 70 kg individuals from mouse experiments.

In some embodiments, the RNA molecule of the invention is delivered in a liposome formulation, lipofectin formulation, or the like and can be prepared by methods well known to those of skill in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972, 5,589,466 and 5,580,859, which are incorporated herein by reference. It is described in. Delivery systems have been developed specifically for the purpose of enhancing and improving siRNA delivery to mammalian cells (eg, Shen et al FEBS Let. 2003, 539: 111-114; Xia et al., Nat. Biotech. 2002. 20: 1006-1010; Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11: 2717-2724), which can be applied to the present invention. SiRNA has been successfully used in the suppression of gene expression in primates in recent years (see, for example, Tolentino et al., Retina 24 (4): 660), which can also be applied to the present invention.

General Information on Particle Delivery In addition, a method of preparing sgRNA / Cas9 protein-containing particles in which a mixture containing sgRNA and Cas9 protein (and optionally an HDR template) is used as a surfactant, phospholipid, biodegradable polymer. , A method comprising the step of containing or mixing with a mixture consisting of or consisting essentially of lipoproteins and alcohols; and for particles from such methods, "targeting disorders and diseases with particle delivery components". CRISPR-CAS system and composition for delivery, use and therapeutic application (DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORGETING DISORGETING DISORDERS / US14 / 70057, Agent Reference No. 47627.99.2060 and BI-2013 / 107 (US Provisional Patent Application No. 62 / 054,490 filed on September 24, 2014; 2014; The same No. 62 / 010,441 filed on June 10, and the same Nos. 61 / 915,118 and 61/915,215 filed on December 12, 2013, respectively. ("Particle Delivery PCT") (incorporated herein by reference), which claims priority from one or more of the specification and the same No. 61 / 915,148. For example, Cas9 protein and sgRNA are preferably in a sterile nuclease-free buffer, such as 1 x PBS, in a preferred, eg, 3: 1-1: 3 or 2: 1-1: 2, or 1: 1 molar. In proportion, they were mixed together at a suitable temperature, such as 15-30 ° C., such as 20-25 ° C., for example room temperature, for a suitable time, such as 15-45, for example 30 minutes. Apart from that, surfactants such as cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); phospholipids such as dimyristoylphosphatidylcholine (DMPC); biodegradable polymers such as ethylene. Glycolpolymers or PEGs and lipoproteins such as low density lipoproteins such as cholesterol, or particle components containing them, are alcohols, preferably C1-6 alkyl alcohols such as methanol, ethanol, isopropanol, such as 100. Dissolved in% ethanol. These two solutions were mixed together to form particles containing the Cas9-sgRNA complex. Therefore, the sgRNA may be pre-formed into a complex with the Cas9 protein before forming the entire complex as particles. Different components of different molar ratios known to facilitate delivery of nucleic acids to cells (eg 1,2-dioleoil-3-trimethylammonium propane (DOTAP), 1,2-ditetradecanoyl-sn- Formulations may be made with glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG), and cholesterol), eg DOTAP: DMPC: PEG: cholesterol molar ratios are DOTAP100, DMPC0, PEG0, cholesterol 0; or DOTAP90, DMPC0, PEG10, cholesterol 0; or DOTAP90, DMPC0, PEG5, cholesterol 5, DOTAP100, DMPC0, PEG0, cholesterol 0. The application therefore includes the step of mixing the sgRNA, the Cas9 protein and the constituents forming the particles; and the particles from such a mixing step. Aspects of the invention are particles; eg, a mixture containing sgRNA and / or Cas9 as in the present invention and a particle-forming component, such as in particle delivery PCT, to form particles. Thus, for example, particles using a method similar to particle delivery PCT and particles from such a mixing step (or, of course, other particles containing sgRNA and / or Cas9 as in the present invention) may be included.

Particle Delivery Systems and / or Formulations Several types of particle delivery systems and / or formulations are known to be useful in a wide range of biomedical applications. Generally, a particle is defined as a small object that behaves as a unit as a whole with respect to its transport and properties. Particles are further classified according to diameter. Coarse particles cover the range of 2,500 to 10,000 nanometers. The fine particles are 100 to 2,500 nanometers in size. Ultrafine particles, or nanoparticles, are generally 1 to 100 nanometers in size. This 100 nm limit is based on the fact that new properties that distinguish particles from bulk materials typically occur on a critical length scale of less than 100 nm.

As used herein, a particle delivery system / formulation is defined as any biological delivery system / formulation that includes particles according to the invention. The particles according to the present invention are any entity having a maximum size (eg, diameter) of less than 100 microns (μm). In some embodiments, the particles of the invention have a maximum size of less than 10 μm. In some embodiments, the particles of the invention have a maximum size of less than 2000 nanometers (nm). In some embodiments, the particles of the invention have a maximum size of less than 1000 nanometers (nm). In some embodiments, the particles of the invention have maximum dimensions of 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or less than 100 nm. Typically, the particles of the invention have a maximum size (eg, diameter) of 500 nm or less. In some embodiments, the particles of the invention have a maximum size (eg, diameter) of 250 nm or less. In some embodiments, the particles of the invention have a maximum size (eg, diameter) of 200 nm or less. In some embodiments, the particles of the invention have a maximum size (eg, diameter) of 150 nm or less. In some embodiments, the particles of the invention have a maximum size (eg, diameter) of 100 nm or less. Smaller particles, such as particles with a maximum size of 50 nm or less, are used in some embodiments of the invention. In some embodiments, the particles of the invention have a maximum size in the range of 25 nm to 200 nm.

Particle characterization (including characterizing, for example, morphology, dimensions, etc.) is performed using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform. Transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization flight time mass analysis (MALDI-TOF), ultraviolet / visible spectroscopy, double polarization interference and nuclear magnetic resonance (NMR). The characterization (dimensioning) may be performed on natural particles (ie, before loading) or to provide particles of optimal size for delivery to any in vitro, ex vivo and / or in vivo application of the invention. Thus, cargo (in the present specification, cargo refers, for example, to one or more components of the CRISPR-Cas system, such as CRISPR enzyme or mRNA or guide RNA, or any combination thereof, additional carriers and / or modifications. It may be done after loading (which may contain agents). In certain preferred embodiments, the characterization of particle size (eg, diameter) is based on measurements using dynamic laser scattering (DLS). Regarding particles, their preparation and use, and their measurement, US Pat. No. 8,709,843; US Pat. No. 6,007,845; US Pat. No. 5,855,913; US Pat. No. 5,985,309; US Pat. No. 5,543,158; and James E. et al. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online 11 May 2014, doi: 10.1038 / nano. See the announcement by 2014.84.

Particle delivery systems within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles. Thus, any delivery system described herein is a particle within the scope of the invention, including, but not limited to, lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene guns. It can be provided as a delivery system.

Particle CRISPR enzyme mRNA and guide RNA can be delivered simultaneously using particles or lipid envelopes; for example, the CRISPR enzyme and RNA of the invention (eg, as a complex), such as 7C1, are described in Dahlman et al. , International Publication No. 2015089419 A2 pamphlet and particles as in its references (eg, James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) online publication 11 May 2014, doi: 10.1038 / non. .84), eg, delivery particles containing lipids or lipidoids and hydrophilic polymers such as cationic lipids and hydrophilic polymers [eg 1,2-dioreoil-3-trimethylammonium propane (DOTAP) for cationic lipids). Or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) is included, and / or hydrophilic polymers include ethylene glycol or polyethylene glycol (PEG); and / or here. The particles further contain cholesterol (eg, formulation 1 = DOTAP 100, DMPC 0, PEG 0, cholesterol 0; formulation number 2 = DOTAP 90, DMPC 0, PEG 10, cholesterol 0; formulation number 3 = DOTAP 90, DMPC 0, Particles from PEG 5, cholesterol 5), where the particles are formed using an efficient multi-step process, where first the effector proteins and RNA are added, eg, in a 1: 1 molar ratio, eg, at room temperature, eg. Mix together for 30 minutes, eg in sterile nuclease-free 1x PBS; and separately, dissolve DOTAP, DMPC, PEG, and cholesterol as applicable to the formulation in alcohol, eg, 100% ethanol; When these two solutions are mixed together, particles containing the complex are formed)].

For example, Su X, Fricke J, Kavanagh DG, Irvine DJ (“In vitro and in vivo mRNA departure-enveloped pH-responic lipid-enveloped pH-responsive polym.Nol8; 10.102 / mp100390w.Epub 2011 Apr 1) describes biodegradable core-shell structural particles with poly (β-aminoester) (PBAE) cores covered by a phospholipid bilayer shell. These have been developed for the delivery of mRNA in vivo. The pH-responsive PBAE component was selected to promote endosome disruption, while the lipid surface layer was selected to minimize the toxicity of the polycation core. Therefore, they are preferred for delivery of the RNA of the present invention.

In one embodiment, particles based on a self-assembling bioadhesive polymer are contemplated, the particles being applied for oral delivery of peptides, which are all delivery to the brain, intravenous delivery of peptides, and nasal delivery of peptides. Can be done. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs, are also contemplated. Molecular envelope techniques include modified polymer envelopes delivered to protected disease sites (eg, Mazza, M. et al. ACSNano, 2013.7 (2): 1016-1026; Seew, A., et al. Mol Pharma, 2012.9 (1): 14-28; Lalatsa, A., et al. J Control Rel, 2012.161 (2): 523-36; Lalatsa, A., et al., Mol Polymer, 2012 .9 (6): 1665-80; Lalatsa, A., et al. Mol Pharma, 2012.9 (6): 1764-74; Garrett, NL, et al. J Biophotonics, 2012.5 (5) -6): 458-68; Garrett, NL, et al. J Raman Spec, 2012.43 (5): 681-688; Ahmad, S., et al. J Royal Envelope 2010.7: S423 -33; Uchegbu, IF Expert Opin Drug Dev, 2006.3 (5): 629-40; Qu, X., et al. Biophotonicules, 2006.7 (12): 3452-9, and Uchegbu, I. F., et al. Int J Polymer, 2001.224: 185-199). A dose of about 5 mg / kg is contemplated and may be a single dose or multiple doses depending on the target tissue.

In one embodiment, particles developed in Dan Anderson's laboratory at MIT that can deliver RNA to cancer cells to stop tumor growth can be used in the CRISPR Cas system of the invention. And / or can be adapted. In particular, Anderson's laboratory has developed a fully automated combination system for the synthesis, purification, characterization, and formulation of new biomaterials and nanoforms. For example, Alabi et al. , Proc Natl Acad Sci US A. 2013 Aug 6; 110 (32): 12881-6; Zhang et al. , Adv Mater. 2013 Sep 6; 25 (33): 4641-5; Jiang et al. , Nano Lett. 2013 Mar 13; 13 (3): 1059-64; Karagiannis et al. , ACS Nano. 2012 Oct 23; 6 (10): 8484-7; Whitehead et al. , ACS Nano. 2012 Aug 28; 6 (8): 6922-9, and Lee et al. , Nat Nanotechnology. See 2012 Jun 3; 7 (6): 389-93.

U.S. Patent Application Publication No. 20110293703 relates to lipid compounds that are also particularly useful for the administration of polynucleotides, which are applicable to the delivery of the CRISPR Cas system of the invention. In one aspect, the aminoalcohol lipid compound is combined with an agent to be delivered to a cell or subject to form microparticles, nanoparticles, liposomes, or micelles. The agent to be delivered by particles, liposomes, or micelles can be in the form of a gas, liquid, or solid, which agent can be a polynucleotide, protein, peptide, or small molecule. This aminoalcohol lipid compound can be combined with other aminoalcohol lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids and the like to form particles. These particles can then be optionally combined with pharmaceutical excipients to form a pharmaceutical composition.

U.S. Patent Application Publication No. 20110293703 also provides a method for preparing aminoalcohol lipid compounds. One or more equivalents of amines are reacted with one or more equivalents of epoxide-terminated compounds under conditions suitable for forming the aminoalcohol lipid compounds of the invention. In certain embodiments, all transaminations of the amine react well with the epoxide-terminated compound to form a tertiary amine. In other embodiments, not all amino groups of the amine react well with the epoxide-terminated compound to form a tertiary amine, thus a primary or secondary amine in the aminoalcohol lipid compound. Is formed. These primary or secondary amines can remain intact or react with other electrophiles, such as different epoxide-terminated compounds. As will be appreciated by those skilled in the art, reacting the amine with less than an excess of epoxide-terminated compounds results in a number of different aminoalcohol lipid compounds with varying numbers of tails. Certain amines can be fully functionalized at the tails of the two epoxide-derived compounds, while other molecules are not sufficiently functionalized at the tails of the epoxide-derived compounds. For example, diamines or polyamines can include one, two, three, or four epoxide-derived compound tails away from the various amino moieties of the molecule, primary amines, secondary amines, and tertiary amines. Amines are formed. In certain embodiments, all amino groups are not completely functional. In certain embodiments, two identical types of epoxide-terminated compounds are used. In other embodiments, two or more different epoxide-terminated compounds are used. The synthesis of aminoalcohol lipid compounds is carried out with or without a solvent, and the synthesis can be carried out at a high temperature of 30-100 ° C, preferably about 50-90 ° C. The prepared aminoalcohol lipid compound can be arbitrarily purified. For example, a mixture of aminoalcohol lipid compounds can be purified to give an aminoalcohol lipid compound with a certain number of epoxide-derived compound tails. Alternatively, the mixture can be purified to give a particular steric or positional isomer. Amino alcohol lipid compounds can also be alkylated with alkyl halides (eg, methyl iodide) or other alkylating agents and / or acylated.

U.S. Patent Application Publication No. 20110293703 also provides a library of aminoalcohol lipid compounds prepared by the methods of the invention. These aminoalcohol lipid compounds can be prepared and / or screened using high throughput techniques including liquid handlers, robots, microtiter plates, computers and the like. In certain embodiments, aminoalcohol lipid compounds are screened for their ability to transfect polynucleotides or other agents (eg, proteins, peptides, small molecules) into the cell.

U.S. Patent Application Publication No. 20130302401 relates to a class of poly (β-aminoalcohol) (PBAA) prepared using combinatorial polymerization. The PBAA of the present invention is a biotechnology as a coating (eg, a thin or multilayer thin film coating for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cell encapsulants. And can be used for medical purposes. When used as a surface coating, these PBAAs caused different levels of inflammation, both in vitro and in vivo, due to their chemical structure. The wide range of chemical varieties of materials in this class allowed the identification of polymer coatings that inhibit macrophage activity in vitro. In addition, these coatings reduce the recruitment of inflammatory cells and reduce fibrosis after subcutaneous injection of carboxylated polystyrene microparticles. These polymers can be used to form polymer electrolyte composite capsules for cell encapsulation. The present invention may also have many other biological applications such as, for example, antibacterial coatings, delivery of DNA or siRNA, and engineering of stem cell tissues. The teachings of US Patent Application Publication No. 20130302401 can be applied to the CRISPR Cas system of the present invention. In some embodiments, as described herein, and with respect to delivery applications to any particle unless otherwise apparent, WO 2014118272 pamphlet (incorporated herein by reference) and Nair, JK. et al. , 2014, Journal of the American Chemical Society 136 (49), 16958-16961) and the teachings herein may be used with sugar-based particles such as GalNAc.

In another embodiment, lipid particles (LNP) are also contemplated. Anti-transthyretin short interfering RNA is encapsulated in lipid particles and delivered to humans (see, eg, Coelho et al., N Engl J Med 2013; 369: 819-29) and such systems. , Can be applied by adapting to the CRISPR Cas system of the present invention. A dose of about 0.01 to about 1 mg per kg body weight administered intravenously is contemplated. Drugs that reduce the risk of infusion-related reactions are contemplated, such as dexamethasone, acetampinophen, diphenhydramine or cetirizine, and ranitidine. Multiple doses of about 0.3 mg / kg every 4 weeks for a total of 5 doses are also planned.

LNP has been shown to be extremely effective in delivering siRNA to the liver (see, eg, Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470). Therefore, delivery of RNA encoding CRISPR Cas to the liver is intended. Approximately 4 doses of 6 mg / kg of LNP every 2 weeks can be planned. Tabernero et al. Observed tumor regression after the first two cycles of 0.7 mg / kg LNP administration, and by the end of the six cycles, patients had complete regression of lymph node metastases and substantial shrinkage of liver tumors. It was demonstrated that a partial reaction including was achieved. A complete response was obtained after 40 doses in this patient, who maintained a remission phase and terminated treatment after 26 months of dosing. Two patients with extrahepatic position and RCC of disease, including kidney, lung, and lymph nodes, advanced after prior treatment with VEGF pathway inhibitors, have stable disease at all sites for approximately 8-12 months Patients with PNET and liver metastasis continued the extended study for 18 months (36 doses) with stable disease.

However, changes in LNP must be considered. Cationic lipids are combined with negatively charged lipids to induce a monolayer structure that facilitates intracellular delivery. Since charged LNPs are rapidly removed from circulation after intravenous injection, ionic cationic lipids with a pKa value of less than 7 have been developed (eg, Rosin et al, Molecular Therapy, vol. 19, no. 12, etc.). See pages 1286-2200, Dec. 2011). A negatively charged polymer, such as RNA, can be introduced into the LNP at a low pH value (eg, pH 4) and the ionic lipid can exhibit a positive charge. However, at physiological pH values, LNPs exhibit low surface charges that are compatible with long circulation times. Four types of ionic cationic lipids, namely 1,2-dilineoil-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N, N-dimethylaminopropane (DLinDMA). , 1,2-Dilinoleyloxyketo-N, N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1, 3] -Focused on dioxolane (DLinKC2-DMA). LNP siRNA systems containing these lipids show significantly different gene silencing properties in hepatocytes in vivo and differ efficacy according to the series DLinKC2-DMA> DLinKDMA> DLinDMA >> DLinDAP utilizing the Factor VII gene silencing model. (See, for example, Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011). In particular, in the case of formulations containing DLinKC2-DMA, a dose of 1 μg / ml of LNP or CRISPR Cas RNA within or associated with this LNP can be contemplated.

In the preparation of LNP and CRISPR Cas encapsulation, Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011 can be used and / or adapted to this document. Cationic lipids, 1,2-diline oil-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleioxy-3-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinoleioxyketo-N , N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1,3] -dioxolane (DLinKC2-DMA), (3-O- [ 2''-(Methoxypolyethylene glycol 2000) succinoyl] -1,2-dimyristyl-sn-glycol (PEG-S-DMG), and R-3-[(ω-methoxy-poly (ethylene glycol) 2000) carbamoyl ] -1,2-Dimyristyloxypropyl-3-amine (PEG-C-DOMG) may be given from Tekmira Pharmaceuticals (Vancover, Canda) or may be synthesized. Cholesterol may be given from Sigma (Vancouver, Canda). It can be purchased from St Louis, MO). Specific CRISPR Cas RNAs are cationic lipids of DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (molar ratio of 40:10:40:10): DSPC :. CHOL: Can be encapsulated in LNP containing (PEGS-DMG or PEG-C-DOMG). If desired, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) can be encapsulated to assess cell uptake, intracellular delivery, and biodistribution. For encapsulation, a lipid mixture consisting of cationic lipid: DSPC: cholesterol: PEG-c-DOMG (molar ratio of 40:10:40:10) is dissolved in ethanol to a final lipid concentration of 10 mmol / l. Can be done by. An ethanol solution of this lipid can be added dropwise to 50 mmol / l citrate, pH 4.0 to form multiple vesicles to a final concentration of 30% ethanol (vol / vol). Large monolayer vesicles can be formed after passing multilayer vesicles through a double 80 nm Nuclepore polycarbonate filter using an extruder (Northern Lipids, Vancouver, Canada). To this extruded, preformed large monolayer vesicle, RNA dissolved in 50 mmol / l citrate, pH 4.0, 2 mg / ml containing 30% ethanol (vol / vol) was added dropwise, and 0 Encapsulation can be achieved by incubating at 31 ° C. for 30 minutes with constant mixing to a final RNA / lipid weight ratio of .06 / 1 wt / wt. Ethanol removal and neutralization of the formulation buffer were performed by phosphate buffered saline (PBS) using a Spectra / Por 2 regenerated cellulose dialysis membrane, dialysis against pH 7.4 for 16 hours. The particle size distribution can be determined by dynamic light scattering and Gaussian fitting using a NICOMP 370 particle sorter (Nicomp Curve Sizing, Santa Barbara, CA) in vesicle / intensity mode. The particle size of all three LNP systems can be about 70 nm in diameter. RNA encapsulation efficiency can be determined by removal of free RNA from samples collected before and after dialysis using a VivaPureD MiniH column (Sartorius Stedim Biotech). Encapsulated RNA can be extracted from the eluted particles and quantified at 260 nm. The ratio of RNA to lipid was determined by measuring the cholesterol content in the parenchyma using the Wako Chemicals USA (Richmond, VA) cholesterol enzyme assay. In connection with the discussion of LNPs and PEG lipids herein, PEGylated liposomes or LNPs are also suitable for delivery of the CRISPR-Cas system or its components.

For the preparation of large LNPs, Rosin et al, Modular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011 can be used and / or adapted to this document. A lipid premix solution (total lipid concentration of 20.4 mg / ml) can be prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol in a molar ratio of 50:10: 38.5. Sodium acetate can be added to the lipid premix in a molar ratio of 0.75: 1 (sodium acetate: DLinKC2-DMA). The lipid can then be hydrated by combining the mixture with 1.85 times the amount of citrate buffer ((10 mmol / l, pH 3.0) with vigorous stirring, thereby 35%. Liposomes form spontaneously in aqueous buffer containing ethanol. The liposome solution can be incubated at 37 ° C. to increase particle size in a time-dependent manner. Aliquots are removed at various times during the incubation. Therefore, changes in liposome size can be evaluated by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once the desired particle size is achieved, an aqueous PEG lipid solution (stock = 35% (vol / vol) in ethanol, 10 mg / ml PEG-DMG) is added to the liposome mixture to finalize 3.5% of total lipids. It can be PEG molar concentration. Upon addition of PEG-lipid, liposomes should effectively stop their size from growing further. RNA can then be added to empty liposomes at a ratio of RNA to total lipids of about 1:10 (wt: wt) and then incubated at 37 ° C. for 30 minutes to form packed LNPs. The mixture can then be dialyzed overnight in PBS and filtered through a 0.45-μm syringe filter.

Physical Nucleic Acid (SNA ™) constructs and other particles (particularly gold particles) are also contemplated as means of delivering the CRISPR-Cas system to the intended target. Significant data indicate that the AuraSense Therapeutics' Physical Nuclear Acid (SNA ™) construct based on nucleic acid-functionalized gold particles is useful.

References that can be used in connection with the teachings herein are: Cutler et al. , J. Am. Chem. Soc. 2011 133: 9254-9257, Hao et al. , Small. 2011 7: 3158-3162, Zhang et al. , ACS Nano. 2011 5: 6962-6970, Cutler et al. , J. Am. Chem. Soc. 2012 134: 1376-391, Young et al. , Nano Lett. 2012 12: 3867-71, Zheng et al. , Proc. Natl. Acad. Sci. USA. 2012 109: 11975-80, Milkin, Nanomedicine 2012 7: 635-638 Zhang et al. , J. Am. Chem. Soc. 2012 134: 16488-1691, Weintraub, Nature 2013 495: S14-S16, Choi et al. , Proc. Natl. Acad. Sci. USA. 2013 110 (19): 7625-7630, Jensen et al. , Sci. Translation. Med. 5,209ra152 (2013), and Milkin, et al. , Small, 10: 186-192.

Self-constructing particles containing RNA can be formed using polyethyleneimine (PEI) PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of polyethylene glycol (PEG). This system, for example, as a means of targeting integrin-expressing tumor neovascularization and delivering siRNA that suppresses the expression of vascular endothelial growth factor receptor-2 (VEGF R2), thereby achieving tumor angiogenesis. Used (see, for example, Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplex can be prepared by mixing an equal amount of an aqueous solution of a cationic polymer and nucleic acid, and applying a net molar excess of ionized nitrogen (polymer) to the phosphate (nucleic acid) in the range of 2 to 6. .. The electrostatic interaction between the cationic polymer and the nucleic acid forms a polyplex with an average particle size distribution of about 100 nm and is therefore referred to herein as a nanoplex. A dose of about 100-200 mg of CRISPR Cas is conceivable for delivery in self-assembled particles of Schiffelers et al.

Nanoplexes by Bartlett et al. (PNAS, September 25, 2007, vol.104, no.39) can also be applied to the present invention. Bartlett et al. Nanoplex is prepared by mixing an equal volume of aqueous solution of a cationic polymer with nucleic acid and adding a net molar excess of ionized nitrogen (polymer) to the phosphate (nucleic acid) in the range 2-6. .. The electrostatic interaction between the cationic polymer and the nucleic acid forms a polyplex with an average particle size distribution of about 100 nm and is therefore referred to herein as a nanoplex. The DOTA-siRNA of Bartlett et al. Was synthesized as follows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate mono (N-hydroxysuccinimide ester) (DOTA-NHTester). Was ordered from Cyclocyclics (Dallas, TX). It was added to a microcentrifuge tube with amine-modified RNA sense strand in carbonate buffer (pH 9) and a 100-fold molar excess of DOTA-NHSester. The contents were reacted by stirring at room temperature for 4 hours. The DOTA-RNA sense strand conjugate was ethanol precipitated, resuspended in water and annealed to the unmodified antisense strand to give DOTA-siRNA. All liquids were treated with Celex-100 (Bio-Rad, Hercules, CA) to remove trace metal contaminants. Tf-targeted siRNA particles and Tf-non-targeted siRNA particles can be formed using cyclodextrin-containing polycations. Typically, the particles were formed in water with a charge ratio of 3 (±) and a siRNA concentration of 0.5 g / liter. 1% adamantane-PEG molecule on the surface of the target particle was modified with Tf (adamantane-PEG-Tf). The particles were suspended in a 5% (wt / vol) glucose carrier solution for injection.

Davis et al. (Nature, Vol 464,15 April 2010) perform RNA clinical trials using a target particle delivery system (clinical trial registration number NCT00689065). Patients with solid tumors who are ineffective with standard cancer therapy are given target particles by intravenous injection for 30 minutes on days 1, 3, 8, and 10 of the 21-day cycle. The particles are: (1) a linear cyclodextrin-based polymer (CDP), (2) human transferrin that targets a ligand presented on the outer surface of the particle to bind to a TF receptor (TFR) on the surface of cancer cells. Designed to suppress the expression of proteins (TF), (3) hydrophilic polymers (polyethylene glycol (PEG) used to improve particle stability in biofluids), and (4) RRM2. It consists of a synthetic delivery system containing siRNA (the sequence used in the clinic has already been shown as siR2B + 5). TFR has long been known to be upregulated in malignant cells, and RRM2 is an established anticancer target. These particles (clinical form shown as CALAA-01) have been shown to be well tolerated in multiple dose studies in non-human primates. Although siRNA was administered by liposome delivery to a patient with chronic myelogenous leukemia, the clinical trial by Davis et al. Was the first human trial to deliver siRNA systemically using a targeted delivery system to treat patients with solid cancer. Is. To determine if the targeted delivery system can effectively deliver functional siRNA to human tumors, Davis et al. Have three patients that make up three different dosing cohorts: each with metastatic melanoma. Biopsies from patients A, B, and C who received the doses of 18, 24, and 30 mg / m ² of CALAA-01, respectively, were examined. Similar doses can be contemplated for the CRISPR Cas system of the invention. The delivery of the present invention is a linear cyclodextrin-based polymer (CDP), a human transferrin protein (TF) that targets a ligand presented on the outer surface of the particle to bind to the TF receptor (TFR) on the surface of cancer cells. ), And / or particles containing a hydrophilic polymer (eg, polyethylene glycol (PEG) used to improve the stability of the particles in a biofluid).

For the present invention, it is preferred to deliver one or more components of the CRISPR complex, such as CRISPR enzyme or mRNA or guide RNA, using particles or lipid envelopes. Other delivery systems or vectors can be used in connection with aspects of the particles of the invention.

In general, a "nanoparticle" is any particle having a diameter of less than 1000 nm. In certain preferred embodiments, the nanoparticles of the invention have a maximum size (eg, diameter) of less than 500 nm. In another preferred embodiment, the nanoparticles of the invention have a maximum size in the range of 25 nm to 200 nm. In another preferred embodiment, the nanoparticles of the invention have a maximum size of less than 100 nm. In another preferred embodiment, the nanoparticles of the invention have a maximum size in the range of 35 nm to 60 nm.

The particles included in the present invention are in various forms, such as solid particles (eg, metals such as silver, gold, iron, titanium), non-metals, lipid-based solids, polymers, suspensions of particles, or. It can be provided as a combination of these. Metals, dielectrics, and semiconductor particles, as well as hybrid structures (eg, core-shell particles) can be prepared. Particles formed from semiconductor materials can also be labeled quantum dots if they are small enough (typically less than 10 nm) to cause quantization of electron energy levels. Such nanoscale particles can be used in biomedical applications as drug carriers or contrast agents and can be adapted for similar purposes of the present invention.

Semi-solid particles and flexible particles are produced, which are within the scope of the present invention. The semi-solid prototype particles are liposomes. Various types of liposome particles are currently used clinically as delivery systems for anticancer agents and vaccines. Particles that are half hydrophilic and the other half hydrophobic are called Janus particles and are particularly effective in stabilizing emulsions. The particles can self-build at the water / oil interface and function as a solid surfactant.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides a drug delivery system for targeted delivery of particles containing a therapeutic agent to histiocytes, cells, and intracellular compartments. The present invention provides target particles containing surfactants, hydrophilic polymers, or lipid-conjugated polymers. US Pat. No. 6,007,845, incorporated herein by reference, is a multiblock formed by covalent bonding of a polyfunctional compound with one or more hydrophobic polymers and one or more hydrophilic polymers. Provided are particles having a copolymer core and containing a biologically active material. U.S. Pat. No. 5,855,913, incorporated herein by reference, has aerodynamically light particles with a tap density of less than 0.4 g / cm3 and an average diameter of 5 μm to 30 μm, and. A fine particle composition containing a surfactant for delivering a drug to the lung system on its surface is provided. US Pat. No. 5,985,309, which is incorporated herein by reference, is a surfactant for delivery to the lung system and / or a charge opposite to that of a positively or negatively charged therapeutic or diagnostic agent. Provided are particles containing a hydrophilic or hydrophobic complex with a charged molecule. US Pat. No. 5,543,158, which is incorporated herein by reference, is a biodegradable solid core having a biodegradable solid core containing a biologically active material and a poly (alkylene glycol) moiety on the surface. Provide particles for injection. International Publication No. 20121235025 (also published as US Patent Application Publication No. 201212551560), incorporated herein by reference, contains conjugated polyethyleneimine (PEI) polymers and conjugated bruises macrocycles (summary). It describes a "conjugated lipomer" or "lipomer"). In certain embodiments, such conjugated lipomers are associated with the CRISPR-Cas system, which achieves genomic perturbations in vivo, ex vivo, and in vitro to regulate gene expression, including regulation of protein expression. It can be assumed that it can be used in relation to. On

In one embodiment, the particles can be an epoxide-modified lipid polymer, advantageously 7C1 (eg, James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) online publication 11 May 2014, doi: 10.1038 / nano. . 2014.84). C71 was synthesized by reacting C15 epoxide-terminated lipid with PEI600 at a molar ratio of 14: 1 and using C14PEG2000 to formulate stable particles (35-60 nm diameter) in PBS solution for at least 40 days. ..

Epoxide-modified lipid-polymers can be utilized to deliver the CRISPR-Cas system of the invention to lung cells, cardiovascular cells, or renal cells, to those skilled in the art for delivery to other target organs. This system could be adapted. Dose of about 0.05 to about 0.6 mg / kg is conceivable. Administration over days or weeks with a total dose of about 2 mg / kg is also conceivable.

Exosomes Exosomes are endogenous nano-vesicles that transport RNA and proteins and can deliver RNA to the brain and other target organs. To reduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnology 29: 341) used autologous dendritic cells to generate exosomes. Targeting the brain was achieved by engineering dendritic cells to express Lamp2b, an exosome membrane protein fused to a neuron-specific RVG peptide. Purified exosomes were added to exogenous RNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA, especially to neurons, microglia, and oligodendrocytes in the brain, resulting in specific gene knockdowns. Pre-exposure to RVG exosomes did not attenuate knockdown and no non-specific uptake into other tissues was observed. The therapeutic potential of exosome-mediated siRNA delivery has been demonstrated by knockdown of the potent mRNA (60%) and protein (62%) of BACE1, a therapeutic target for Alzheimer's disease.

To obtain a pool of immunologically inactive exosomes, Alvarez-Erviti et al. Collected bone marrow from allogeneic major histocompatibility complex (MHC) haplotype inbred C57BL / 6 mice. Because immature dendritic cells produce large amounts of exosomes free of T cell activators such as MHC-II and CD86, Alvarez-Erviti et al. Have been working on granulocyte / macrophage colony stimulating factor (GM- Dendritic cells were selected using CSF). The next day, exosomes were purified from the culture supernatant using a well-established ultracentrifugation protocol. The resulting exosomes were physically homogeneous and the size distribution peaked at a diameter of 80 nm as determined by particle tracking analysis (NTA) and electron microscopy. Alvarez-Erviti We obtained exosomes 10 ⁶ cells per 6～12Myug (measured based on the protein concentration).

Next, Alvarez-Erviti et al. Investigated the possibility of introducing exogenous cargo into modified exosomes using an electroporation protocol adapted to nanoscale applications. Since electroporation of membrane particles on the nanometer scale has not been fully characterized, non-specific Cy5-labeled RNA was used for empirical optimization of electroporation protocols. The amount of RNA encapsulated was analyzed after ultracentrifugation and lysis of exosomes. RNA was maximally retained by electroporation at 400 V and 125 μF and was used in all subsequent experiments.

Alvarez-Erviti et al. Administered 150 μg of each BACE1 siRNA encapsulated in 150 μg of RVG exosomes to normal C57BL / 6 mice and knocked down efficiency in 4 controls: untreated mice, only RVG exosomes were injected. A complex was formed with mice, mice injected with BACE1 siRNA complexed with in vivo cationic liposome reagents, and RVG-9R, an RVG peptide conjugated to 9D-arginine that electrostatically binds to siRNA. BACE1 siRNA was compared to injected mice. Cortical tissue samples were analyzed 3 days after dosing and significant protein knockdown (45%, P <0.05, vs. 62%, P <0) in both siRNA-RVG-9R treated and siRNARVG exosome treated mice. .01) was observed, which was a significant decrease in BACE1 mRNA levels (66% [+ or-] 15%, P <0.001 and 61% [+ or-] 13%, P <0.01, respectively. ). In addition, Applicants have significantly reduced levels of total [β] -amyloid 1-42, which are the main components of amyloid plaque in the pathology of Alzheimer's disease, in RVG-exosome-treated animals (55%, P <0. 05) was demonstrated. The observed reduction was greater than the reduction in β-amyloid 1-40 demonstrated in normal mice after intraventricular injection of the BCAE1 inhibitor. Alvarez-Erviti et al. Performed 5'rapid amplification of the cDNA terminal (RACE) in the BCAE1 cleavage product to provide evidence of RNAi-mediated knockdown by siRNA.

Finally, Alvarez-Erviti et al. Investigated whether RNA-RVG exosomes induced an immune response in vivo by assessing serum concentrations of IL-6, IP-10, TNFα, and IFN-α. .. After exosome treatment, non-significant changes in all cytokines were recorded similar to siRNA transfection reagent treatment in contrast to siRNA-RVG-9R, which strongly stimulates IL-6 secretion, and immunology of exosome treatment. An inactive profile was confirmed. Assuming that the exosome encapsulates only 20% of the siRNA, delivery in the RVG exosome is achieved with equivalent mRNA knockdown and larger protein knockdown with 1/5 siRNA without corresponding levels of immune stimulation. Therefore, it seems to be more efficient than RVG-9R delivery. This experiment demonstrates the therapeutic potential of RVG exosome technology, which may be suitable for long-term silencing of genes associated with neurodegenerative diseases. The exosome delivery system of Alvarez-Erviti et al. Can be applied for delivery of the CRISPR-Cas system of the present invention to therapeutic targets, particularly neurodegenerative diseases. In the present invention, a dose of about 100-1000 mg of CRISPR Cas encapsulated in about 100-1000 mg of RVG exosomes can be contemplated.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126 (2012)) disclose how cultured cell-derived exosomes can be used for RNA delivery in vitro and in vivo. This protocol first describes the production of target exosomes by transfection of an expression vector containing an exosome protein fused to a peptide ligand. Next, El-Andaloussi et al. Describe methods for purification and characterization of exosomes from the supernatant of transfected cells. Next, El-Andaloussi et al. Detailed the important steps of introducing RNA into exosomes. Finally, El-Andaloussi et al. Outline how exosomes are used to efficiently deliver RNA to the mouse brain in vitro and in vivo. Examples of expected results in which exosome-mediated RNA delivery is evaluated by functional assays and imaging are also shown. The entire protocol takes about 3 weeks. Delivery or administration according to the present invention can be carried out using exosomes produced from autologous dendritic cells. From the teachings herein, this can be utilized in the practice of the present invention.

In another embodiment, plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomes are nano-sized vesicles (30-90 nm in size) produced in many cell types, including dendritic cells (DCs), B cells, T cells, mast cells, epithelial cells, and tumor cells. These vesicles are formed by inward budding of late endosomes and then released into the extracellular environment upon fusion with the plasma membrane. Since exosomes naturally transport RNA between cells, this property can be useful for gene therapy, and this disclosure can be utilized in the practice of the present invention.

Exosomes from plasma separate the plasma by centrifugation of the buffy coat at 900 g for 20 minutes, and collect the cell supernatant, remove the cells by centrifugation at 300 g for 10 minutes, and centrifuge at 16500 g for 30 minutes. It can be prepared by separating and then filtering through a 0.22 mm filter. Exosomes are pelleted by ultracentrifugation at 120,000 g for 70 minutes. Chemical transfection of siRNA into exosomes is performed according to the manufacturer's instructions for the RNAi Human / Mouse Starter Kit (Qiagen, Hilden, Germany). SiRNA is added to 100 ml PBS to a final concentration of 2 mmol / ml. After the addition of the HiPerFect transfection reagent, the mixture is incubated at RT for 10 minutes. Exosomes are reisolated using aldehyde / phosphate latex beads to remove excess micelles. Chemical transfection of CRISPR Cas into exosomes can be performed similar to siRNA. Exosomes can be cultured with monocytes and lymphocytes isolated from the peripheral blood of healthy donors. Therefore, it can be contemplated that exosomes containing CRISPR Cas can be introduced into monocytes and lymphocytes and self-reintroduced into humans. Therefore, delivery or administration according to the present invention can be carried out using plasma exosomes.

Liposomes Delivery or administration according to the present invention can be performed on liposomes. Liposomes are spherical vesicle structures composed of a monomembrane or multimembrane lipid bilayer and a relatively impermeable outer lipophilic phospholipid bilayer that surrounds the inner aqueous compartment. Liposomes are biocompatible and non-toxic, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes and allowing their fillers to pass through biological membranes. It has received considerable attention as a drug delivery carrier for transport to the blood-brain barrier (BBB) (eg, Succi and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10). . 1155/2011/46967 (see).

Liposomes can be formed from several different types of lipids; phospholipids are most often used to form liposomes as drug carriers. Liposomal formation occurs naturally when the lipid membrane is mixed with aqueous solution, but can also be facilitated by applying force in the form of shake by using a homogenizer, sonicator, or extruder (eg,). , Succi and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011/469679 (see).

Several other additives can be added to the liposomes to alter the structure and properties of the liposomes. For example, either cholesterol or sphingomyelin can be added to the liposome mixture in order to stabilize the liposome structure and prevent leakage of cargo inside the liposome. In addition, liposomes were prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and disetylphosphate, the average vesicle size of which was adjusted to about 50-100 nm (eg, Succi and Navarro, Journal of Drug Delivery, vol. . 2011, Article ID 469679, 12 liposomes, 2011. doi: 10.1155 / 2011/469679 (see for reference).

Liposomal formulations may consist primarily of natural phospholipids and lipids, such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialogangliosides. Since this formulation is prepared solely from phospholipids, liposome formulations face a number of challenges, one of which is plasma instability. Several attempts have been made to overcome these challenges, especially the treatment of lipid membranes. One of these attempts focused on the treatment of cholesterol. Addition of cholesterol to conventional formulations reduces the rapid release of encapsulated bioactive compounds into plasma, or 1,2-dioreoil-sn-glycero-3-phosphaethanolamine (DOPE) is stable. (See, for example, Speech and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011/469679 (see)).

In certain advantageous embodiments, Trojan Horse liposomes (also known as molecular Trojan horses) are preferred and the protocol is http: // cshprotocols. cshlp. org / content / 2010/4 / pdb. prot5407. It can be confirmed by long. These particles can deliver the transgene throughout the brain after vascular injection. Neutral lipid particles with specific antibodies conjugated to the surface may, but are not limited to, cross the blood-brain barrier by endocytosis. Applicants hypothesize that Trojan horse liposomes will be used to deliver the CRISPR family of nucleases to the brain by vascular injection, which allows whole-brain transgenic animals without fetal manipulation. In vivo administration of about 1-5 g of DNA or RNA in liposomes can be contemplated.

In another embodiment, the CRISPR Cas system can be administered in liposomes, eg, stable nucleic acid lipid particles (SNALP) (eg, Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Please refer to). Daily intravenous injections of approximately 1 mg / kg / day, 3 mg / kg / day, or 5 mg / kg / day of the particular CRISPR Cas targeted in SNALP are contemplated. Daily treatment can be performed for about 3 days, followed by weekly administration for 5 weeks. In another embodiment, specific CRISPR Cas encapsulated in SNALP administered by intravenous injection at a dose of about 1 mg / kg or 2.5 mg / kg is also contemplated (eg, Zimmerman et al., Nature Letters, Vol. .441,4 May 2006). SNALP preparations are lipid 3-N-[(w methoxypoly (ethylene glycol) 2000) carbamoyl] -1,2-dimylristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N. , N-Dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol may be included in a molar percent ratio of 2:40:10:48 (eg). , Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic acid lipid particles (SNALP) have demonstrated effective molecular delivery in highly angiogenic HepG2-derived liver tumors, but poorly angiogenic HCT-116-derived liver tumors. Was not proven (see, eg, Li, Gene Therapy (2012) 19,775-780). SNALP liposomes have a 25: 1 lipid / siRNA ratio and a 48/40/10/2 molar ratio of cholesterol / D-Lin-DMA / DSPC / PEG-C-DMA for D-Lin-DMA and PEG-C- DMA can be prepared by formulating distearoyl phosphatidylcholine (DSPC), cholesterol, and siRNA. The SNALP liposomes obtained are about 80-100 nm in size.

In yet another embodiment, SNALP is a synthetic cholesterol (Sigma-Aldrich, St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3-N-[(w-methoxypoly (w-methoxypoly)). Ethylene glycol) 2000) carbamoyl] -1,2-dimirestyloxypropylamine, and cationic 1,2-dilinoleoyloxy-3-N, N dimethylaminopropane (eg, Geisbert et al., See Lancet 2010; 375: 1896-905). For example, as a bolus intravenous infusion, a total CRISPR Cas at a dose of about 2 mg / kg per dose can be contemplated.

In yet another embodiment, SNALPs are synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-CDMA, and 1,2. -Dilinoleyloxy-3- (N; N-dimethyl) aminopropane (DLinDMA) may be included (see, eg, Judge, J. Clin. Invest. 119: 661-673 (2009)). Formulations used for in vivo studies can have a final lipid / RNA mass ratio of approximately 9: 1.

The safety profile of RNAi nanopharmaceuticals was reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, eg, Advanced Drug Delivery Reviews 64 (2012) 1730-1737). Stable Nucleic Acid Lipid Particles (SNALP) are composed of four different lipids-low pH cationic ionic lipids (DLinDMA), neutral helper lipids, cholesterol, and diffusible polyethylene glycol (PEG) -lipids. The particles have a diameter of about 80 nm and are neutrally charged at physiological pH. In the formulation, ionic lipids play a role in condensing lipids with anionic RNA during particle formation. When positively charged under increasingly acidic endosomal conditions, ionic lipids also mediate the fusion of SNALP to endosome membranes, allowing RNA to be released into the cytoplasm. PEG-lipids later provide a neutral, hydrophilic exterior that stabilizes the particles, reduces agglutination in the formulation, and improves pharmacokinetic properties.

To date, two clinical programs have been initiated using SNALP preparations containing RNA. Tekmira Pharmaceuticals has recently completed a phase I single dose study of SNALP-ApoB in adult volunteers with high LDL cholesterol. ApoB is expressed primarily in the liver and jejunum and is essential for the construction and secretion of VLDL and LDL. Seventeen subjects received a single dose of SNALP-ApoB (dose increase at 7 dose levels). No hepatic toxicity (predicted as potential dose-limiting toxicity based on preclinical studies) was observed. One subject (of two) at the highest dose showed influenza-like symptoms consistent with stimulation of the immune system, and a decision was made to conclude this study.

Alnylam Pharmaceuticals similarly advanced ALN-TTR01. ALN-TTR01 utilizes the SNALP technique described above to treat TTR amyloidosis (ATTR) by targeting both mutant and wild-type hepatocyte production. Three ATTR symptoms have been described: familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) -both caused by autosomal dominant mutations in TTR; and senile caused by wild-type TTR. Systemic amyloidosis (SSA). Recently, a placebo-controlled, single-dose increased phase I study of ALN-TTR01 was completed in patients with ATR. ALN-TTR01 was administered as a 15-minute intravenous infusion to 31 patients (23 study drugs and 8 placebo) in a dose range of 0.01-1.0 mg / kg (based on siRNA). .. Treatment showed good tolerability with no significant increase in liver function tests. Injection-related reactions were above 0.4 mg / kg and were seen in 3 of 23 patients; all responded to reduced infusion rates and all continued the study. Minimal and transient elevations of the serum cytokines IL-6, IP-10, and IL-1ra were seen in 2 patients at the highest dose of 1 mg / kg (preclinical and NHP trials predicted). Was done). Due to the decrease in serum TTR, the expected pharmacodynamic effect of ALN-TTR01 was observed at 1 mg / kg.

In yet another embodiment, SNALP is performed by solubilizing cationic lipids, DSPC, cholesterol, and PEG-lipids, respectively, at a molar ratio of, for example, 40:10:40:10, eg, ethanol. (See Simple et al., Nature Niotechnology, Volume 28 Number 2 Efficiency 2010, pp. 172-177). This lipid mixture was added to aqueous buffer (50 mM citrate, pH 4) and mixed to bring the final ethanol and lipid concentrations to 30% (vol / vol) and 6.1 mg / ml, respectively, 22 before extrusion. Equilibrated at ° C. for 2 minutes. This hydrated lipid was applied to a two-layer filter (Nuclepore) having a pore size of 80 nm at 22 ° C. using a Lipex Extruder (Northern Lipids) until a vesicle diameter of 70 to 90 nm determined by dynamic light scattering analysis was obtained. Extruded through. This generally requires 1 to 3 passes. SiRNA (solubilized in 50 mM citrate, pH 4 aqueous solution containing 30% ethanol) was added to pre-equilibrated (35 ° C.) vesicles at a rate of about 5 ml / min with mixing. When the final target siRNA / lipid ratio of 0.06 (wt / wt) was reached, the mixture was incubated at 35 ° C. for an additional 30 minutes to reconstitute vesicles and encapsulate siRNA. Ethanol was then removed and the external buffer was replaced with PBS (155 mM NaCl, 3 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , pH 7.5) by dialysis or tangential flow dialysis filtration. The siRNA was encapsulated in SNALP using a controlled serial dilution process. The lipid component of KC2-SNALP is DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine (DPPC) used in a molar ratio of 57.1: 7.1: 34.3: 1.4. ), Synthetic lipid (Sigma), and PEG-C-DMA. Once the encapsulated particles were formed, SNALP was dialyzed against PBS and sterilized through a 0.2 μm filter prior to use. The average particle size was 75-85 nm and 90-95% of the siRNA was encapsulated within the lipid particles. The final siRNA / lipid ratio in the formulation used for the in vivo test was about 0.15 (wt / wt). The LNP-siRNA system containing Factor VII siRNA was diluted with sterile PBS to the appropriate concentration immediately prior to use and the formulation was intravenously administered to the lateral tail vein in a total volume of 10 ml / kg. This method and delivery systems thereof can be extrapolated to the CRISPR Cas system of the present invention.

Other Lipids Other cationic lipids, such as the aminolipid 2,2-dilinoleyl-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-KC2-DMA), are CRISPR Cas or a component thereof, or eg, , SiRNA-like nucleic acid molecules encoding this (see, eg, Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and thus the book. It can be used to carry out the invention. Preformed vesicles containing the following lipid compositions can be contemplated: aminolipids in molar ratios of 40/10/40/10, respectively, distearoylphosphatidylcholine (DSPC), cholesterol, and (R) -2,3 bis. Octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propyl carbamate (PEG-lipid), and FVII siRNA / total lipid ratio of about 0.05 (w / w). Particles are squeezed up to 3 times on an 80 nm membrane prior to CRISPR Cas RNA for a narrow particle size distribution of 70-90 nm and a low polydispersity index of 0.11 ± 0.04 (n = 56). Can be extruded. Particles containing the highly potent aminolipid 16 can be used with four lipid components 16, DSPC, cholesterol, and PEG-lipid in a molar ratio of (50/10/38.5 / 1.5). The molar ratio can be further optimized to promote in vivo activity.

Michael S D Kormann et al. The use of lipid envelopes is also preferred in the present invention.

In another embodiment, lipids can be formulated with the CRISPR Cas system of the invention to form lipid particles (LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200, and co-lipid disteroylphosphatidylcholine, cholesterol, and PEG-DMG instead of siRNA using natural vesicle formation procedures It can be formulated in the CRISPR Cas system (see, eg, Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4; doi: 10.1038 / mtna. 2011.3.). The component molar ratio can be about 50/10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / disteroylphosphatidylcholine / cholesterol / PEG-DMG). The final lipid: siRNA weight ratio can be approximately 12: 1 and 9: 1 for DLin-KC2-DMA and C12-200 lipid particles (LNP), respectively. The formulation can have an average particle diameter of about 80 nm with an encapsulation efficiency greater than 90%. A dose of 3 mg / kg can be contemplated.

Tekmira is a portfolio of approximately 95 compatible patents in the United States and abroad related to various aspects of LNP and LNP formulations, all of which can be used and / or adapted to the present invention (eg, United States). Patent No. 7,982,027, No. 7,799,565, No. 8,058,069, No. 8,283,333, No. 7,901, 708, 7,745,651, 7,803,397, 8,101,741 and 8,188,263, the same, the same. 7,915,399, 8,236,943, and 7,838,658, and European Patents 1766035, 1519714, the same. 1781593 and 1664316).

The CRISPR Cas system or its constituents, or nucleic acid molecules encoding them, can be encapsulated and delivered in PLGA microspheres, which can be delivered, for example, as proteins, protein precursors, or partially or completely. US Patent Application Publication Nos. 20130252281, 20130245107, and 20130244279 relating to aspects of the formulation of compositions comprising modified nucleic acid molecules capable of encoding a protein or protein precursor in a processed form. PLGA microspheres detailed in the book (transferred to Moderna Proteins). This formulation can have a molar ratio of 50:10: 38.5: 1.5-3.0 (cationic lipid: fusion lipid: cholesterol: PEG lipid). The PEG lipid can be selected from, but not limited to, PEG-c-DOMG, PEG-DMG. The fusion lipid can be DSPC. In addition, Schrum et al. , Delivery and Formulation of Engineered Nucleic Acids, US Patent Application Publication No. 20120251618.

Nanomerics technology addresses bioavailability challenges in a wide range of therapies, including low molecular weight hydrophobic drugs, peptides, and nucleic acid-based therapies (plasmids, siRNAs, miRNAs). Specific routes of administration for which this technique has demonstrated clear advantages include oral routes, transport across the blood-brain barrier, delivery to solid tumors, and the eye. For example, Mazza et al. , 2013, ACS Nano. 2013 Feb 26; 7 (2): 1016-26; Uchegbu and Seew, 2013, J Pharm Sci. 102 (2): 305-10, and Lalatsa et al. , 2012, J Control Release. See 2012 Jul 20; 161 (2): 523-36.

US Patent Application Publication No. 2005019923 describes a cationic dendrimer for delivering bioactive molecules such as polynucleotide molecules, peptides and polypeptides, and / or pharmaceuticals to the mammalian body. Dendrimers are suitable, for example, for the purpose of delivering bioactive molecules to the liver, spleen, lungs, kidneys, or heart (and even the brain). A dendrimer is a synthetic three-dimensional macromolecule synthesized stepwise from a single branched monomeric unit, the properties and functionality of which can be easily controlled and altered. Dendrimers are synthesized by iteratively adding building blocks to the multifunctional core (divergent approach to synthesis) or towards the multifunctional core (convergent approach to synthesis), where the building blocks are three-dimensional shells. Each time it is added to, a higher generation dendrimer is formed. Polypropylene imine dendrimers start with a diaminobutane core and double-Michael addition of acrylonitrile to a primary amine adds a double amount of amino groups to the diaminobutane, followed by hydrogenation of the nitrile. As a result, the number of amino groups is doubled. Polypropylene imine dendrimers contain 100% protonateable nitrogen and up to 64 terminal amino groups (generation 5, DAB64). Protonable groups are usually amino groups that can accept protons at neutral pH. The use of dendrimers as gene delivery agents is largely focused on the use of polyamide amine and phosphorus-containing compounds with amine / amide mixtures or NP (O ₂ ) S as bonding units, respectively, but in the lower generations. The use of polypropylene immine dendrimers in gene delivery has not been reported. Polypropylene imine dendrimers have also been studied as a pH sensitive release system for drug delivery and as a pH sensitive release system for encapsulation of guest molecules chemically modified with peripheral amino acid groups. Cytotoxicity and the interaction of DNA with polypropylene imine dendrimers, as well as the transfection efficacy of DAB64, have also been studied.

Contrary to previous reports, U.S. Patent Application Publication No. 2005019923 states that cationic dendrimers, such as polypropylene imine dendrimers, are suitable for use in the delivery of bioactive molecules, such as genetic material, to targets. It is based on the finding that it exhibits properties such as specific targeting and low toxicity. In addition, derivatives of cationic dendrimers also exhibit suitable properties for delivery of bioactive molecules to the target. See also bioactive polymers in US Patent Application Publication No. 20080267903, which disclose a variety of polymers, including cationic polyamine polymers and dendrimer polymers. This bioactive polymer has been shown to have antiproliferative activity and is therefore characterized by disorders characterized by unwanted cell proliferation, such as neoplasms and tumors, inflammatory disorders (including autoimmune disorders), psoriasis, and atherosclerosis. It may be useful in the treatment of sexual arteriosclerosis. These polymers can be used alone as activators or as delivery vehicles for other therapeutic agents, such as drug molecules or nucleic acids for gene therapy. In such cases, the inherent antitumor activity of the polymer itself may complement the activity of the agent to be delivered. The disclosure of these patent gazettes, along with the teachings herein, can be used to deliver the CRISPR Cas system or its constituents, or nucleic acid molecules encoding them.

Supercharged protein
Supercharged proteins are a class of engineered or intrinsically disordered proteins with an unusually high positive or negative net theoretical charge and are utilized for the delivery of the CRISPR Cas system or its constituents, or nucleic acid molecules encoding them. Can be done. Both positively or negatively overcharged proteins exhibit excellent ability to withstand thermally or chemically induced aggregation. Positively overcharged proteins can also enter mammalian cells. Cargos that bind to these proteins, such as plasmids, DNA, RNA, or other proteins, may allow the functional delivery of these macromolecules to mammalian cells, both in vitro and in vivo. David Liu's laboratory reported the production and characterization of supercharged proteins in 2007 (Lawence et al., 2007, Journal of the American Chemical Society 129, 10110-10112).

Non-viral delivery of RNA and plasmid DNA to mammalian cells is of value for both research and therapeutic applications (Akinc et al., 2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or other positively overcharged protein) is mixed with RNA in a suitable serum-free medium to allow complex formation before cell addition. Serum inclusion at this stage inhibits the formation of supercharged protein-RNA complexes and reduces the effectiveness of the treatment. The following protocols have been found to be effective against a variety of cell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106, 6111-6116). However, pilot experiments with varying doses of protein and RNA should be performed to optimize procedures for specific cell lines.
(1) on the day before treatment, plating the 1 × 10 ⁵ cells in each well of a 48 well plate.
(2) On the day of treatment, the purified +36 GFP protein is diluted with serum-free medium to a final concentration of 200 nM. RNA is added to a final concentration of 50 nM. Vortex, mix and incubate for 10 minutes at room temperature.
(3) During incubation, the medium is aspirated from the cells and washed once with PBS.
(4) After incubation of +36 GFP and RNA, the protein-RNA complex is added to the cells.
(5) Incubate the cells with the complex at 37 ° C. for 4 hours.
(6) After incubation, the medium is aspirated and washed 3 times with 20 U / mL heparin PBS. Cells are incubated in serum-containing medium for an additional 48 hours, or longer depending on the activity assay.
(7) Analyze cells by immunoblotting, qPCR, phenotypic assay, or other suitable method.

David Liu's lab has further found that +36 GFP is an effective plasmid delivery reagent in a variety of cells. Since plasmid DNA is a larger cargo than siRNA, a proportionally higher +36 GFP protein is required to effectively complex the plasmid. For effective plasmid delivery, Applicants have developed a variant of the +36 GFP protein with a C-terminal HA2 peptide tag, a known endosomal disruptive peptide derived from the influenza virus hemagglutinin protein. Although the following protocols are effective for a variety of cells, it is recommended that the doses of plasmid DNA and supercharged proteins be optimized for specific cell lines and delivery applications as described above.
(1) on the day before treatment, plating the 1 × 10 ⁵ cells in each well of a 48 well plate.
(2) On the day of treatment, the purified p36GFP protein is diluted with serum-free medium to a final concentration of 2 mM. Add 1 mg of plasmid DNA. Vortex, mix and incubate for 10 minutes at room temperature.
(3) During incubation, the medium is aspirated from the cells and washed once with PBS.
(4) After incubation of p36GFP protein and plasmid DNA, the protein-DNA complex is gently added to the cells.
(5) Incubate the cells with the complex at 37 ° C. for 4 hours.
(6) After incubation, the medium is aspirated and washed with PBS. Cells are incubated in serum-containing medium for an additional 24-48 hours.
(7) As appropriate, plasmid delivery is analyzed (eg, by plasmid-driven gene expression). Also, for example, McNaughton et al. , Proc. Natl. Acad. Sci. USA 106, 6111-6116 (2009); Cronican et al. , ACS Chemical Biology 5,747-752 (2010); Colonican et al. , Chemistry & Biology 18, 833-838 (2011); Thompson et al. , Methods in Enzymology 503, 293-319 (2012); Thomasson, D. et al. B. , Et al. , Chemistry & Biology 19 (7), 831-843 (2012). The method of supercharged proteins can be used and / or adapted for delivery of the CRISPR Cas system of the invention. Dr. These systems of Lii and the literature herein, along with the teachings herein, can be used to deliver the CRISPR Cas system or its constituents, or nucleic acid molecules encoding them.

Cell Permeable Peptide (CPP)
In yet another embodiment, a cell permeable peptide (CPP) is contemplated for delivery of the CRISPR Cas system. CPP is a short-chain peptide that promotes cellular uptake of various molecular cargoes (from nanosized particles to small chemical and large DNA fragments). The term "cargo" as used herein, but not limited to, therapeutic agents, diagnostic probes, peptides, nucleic acids, antisense oligonucleotides, plasmids, proteins, particles, liposomes, chromophores, small molecules and radioactivity. Includes groups of substances. In aspects of the invention, the cargo may also include any component of the CRISPR Cas system or the entire functional CRISPR Cas system. Aspects of the invention further provide a method of delivering the desired cargo to the subject, the method comprising: (a) preparing a complex comprising the cell-permeable peptide of the invention and the desired cargo. b) Orally, intra-articularly, intraperitoneally, intrathecally, intra-arterially, intranasally, parenchymal, subcutaneously, intramuscularly, intravenously to the complex. Includes steps of percutaneous, intrarectal, or topical administration. Cargo associates with peptides via covalent chemical linkages or non-covalent interactions.

The function of CPP is to deliver cargo to cells, a process that generally occurs through endocytosis in cargo delivered to the endosomes of living mammalian cells. Cell-permeable peptides vary in size, amino acid sequence, and charge, but CPPs all have one distinction of their ability to translocate cell membranes and facilitate delivery of various molecular cargoes to the cytoplasm or organelles. It has various characteristics. CPP translocation can be divided into three major invasion mechanisms: direct invasion in the membrane, endocytosis-mediated invasion, and translocation by forming transient structures. CPP has been found to have numerous applications in medicine as a drug delivery agent in the treatment of various diseases including cancer and virus inhibitors, and as a contrast agent for cell labeling. Examples of contrast agents include acting as a carrier for GFP, an MRI contrast agent, or quantum dots. CPP has great potential for research and pharmaceutical use as an in vitro and in vivo delivery vector. CPPs typically contain a high relative abundance of positively charged amino acids such as lysine or arginine, or have an amino acid composition having a sequence containing alternating patterns of polar / charged amino acids with non-polar, hydrophobic amino acids. Has. These two types of structures are referred to as polycationic or amphipathic, respectively. The third CPP class is a hydrophobic peptide, which has a low net charge and contains only non-polar residues, or has a hydrophobic amino acid group that is critical for cell uptake. One of the first CPPs to be discovered was the transactivated transcriptional activator (Tat) from human immunodeficiency virus type 1 (HIV-1), which is efficient from the surrounding medium by multiple cell types in culture. It was found to be incorporated into. Since then, the number of known CPPs has increased significantly, creating small molecule synthetic analogs with more effective protein transduction properties. CPPs include, but are not limited to, penetratin, Tat (48-60), transportan, and (R-AhX-R4) (Ahx = aminohexanoyl).

U.S. Pat. No. 8,372,951 provides a CPP derived from an eosinophil cationic protein (ECP) that exhibits high cell permeation efficiency and low toxicity. Also provided is an embodiment of delivering CPP to a vertebrate subject with its cargo. Further embodiments of CPP and its service are described in US Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPP can be used for delivery of the CRISPR-Cas system or its components. The ability to deliver the CRISPR-Cas system or its constituents using CPP is also incorporated by reference as a whole by reference to Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, et al. Genome Res. 2014 Apr 2. [Epub ahead of print] article "Gene disruption by cell-penterating peptide-mediated delivery of Cas9 protein provided here" Cas9 protein from Cas9 protein and guide RNA. It has been demonstrated that treatment with CPP-conjugated recombinant Cas9 protein and CPP complexed guide RNA causes endogenous gene disruption in human cell lines. In this paper, the Cas9 protein was conjugated to CPP via a thioether bond, while the guide RNA was complexed with CPP to form condensed positively charged particles. Genes are efficiently disrupted by simultaneously and sequentially treating human cells, including embryonic stem cells, cutaneous fibroblasts, HEK293T cells, HeLa cells, and embryonic cancer tumor cells, with modified Cas9 and guide RNA. It was shown that off-target mutations were reduced compared to plasmid transfection.

Implantable Device In another embodiment, an implantable device for delivery of the CRISPR Cas system or its constituents, or nucleic acid molecules encoding them, is also contemplated. For example, U.S. Patent Application Publication No. 20110195123 discloses an implantable medical device that locally elutes a drug over a long period of time, the disclosure of which includes several types of such devices, implementations. Treatment modes and methods of implantation are included. The device is composed of a polymeric substance used as its body, eg, a matrix, and a drug, and optionally additional scaffolding material, eg, a metal or additional polymer, and a material that facilitates visibility and imaging. To. Implantable delivery devices can be advantageous for local long-term release, where the drug is applied to the affected area, eg, tumor, inflammation, degenerative extracellular matrix (ECM), or symptom relief. It is released directly to the damaged smooth muscle cells or for prevention. One type of drug is the RNA disclosed above, which system can be used and / or adapted to the CRISPR Cas system of the present invention. The mode of implantation in some embodiments is an existing implantation procedure for other procedures, including proximity irradiation therapy and needle biopsy, which has been recently developed and used. In such cases, the dimensions of the new implant described in the present invention are similar to those of the original implant. Typically, several devices are implanted during the same procedure.

In U.S. Patent Application Publication No. 20110195123, drug delivery implantable or insertable systems, including systems applicable to cavities, eg, peritoneum, and / or biostability, which may optionally be a matrix. Other types of administration are provided, including / or degradable and / or bioabsorbable polymer substrates, to which the drug delivery system is not fixed or attached. Note that the word "insertion" also includes implantation. The drug delivery system is preferably implemented as the "Loder" as described in US Patent Application Publication No. 20110195123.

One polymer or multiple polymers are biocompatible and contain one and / or multiple agents, allowing release of the agent at a controlled rate, and in some embodiments, For example, the total amount of the polymeric substrate, such as the matrix, is optionally and preferably less than or equal to the maximum amount that allows the therapeutic level of the agent to be reached. As a non-limiting example, such an amount is preferably in the range of 0.1 m ^{3 to} 1000 mm ³ , depending on the amount of agent introduced. The Loder can be optionally oversized if incorporated into a device sized, for example, by functionality, such as, but not limited to, the knee joint and intrauterine or cervical ring.

The drug delivery system (for composition delivery) is designed to utilize degradable polymers in some embodiments, and the main release mechanism is bulk erosion; or in some embodiments, Non-degradable or slowly degrading polymers are used and the main release mechanism is diffusion rather than bulk erosion, so that the outside acts as a membrane and the inside is long-term (eg, about 1 week ~ It functions as a drug storage that is not actually affected by the surroundings for several months). Combinations of different polymers with different release mechanisms can also be optionally used. The concentration gradient on the surface is preferably kept virtually constant for a significant period of the total drug release period, thus the diffusion rate is virtually constant (referred to as "0 mode" diffusion). The term "constant" preferably means a diffusion rate maintained above the lower threshold of therapeutic effect, but may still optionally have the characteristics of an initial burst and / or, for example, to some extent. It can fluctuate by increasing or decreasing. The diffusion rate is preferably maintained in this way over a long period of time and can be considered constant to a certain level in order to optimize the therapeutic efficacy period, eg, the effective silencing period.

The drug delivery system is designed to protect nucleotide-based therapeutics from degradation, optionally and preferably by chemical properties or by attack from enzymes and other factors in the subject's body. ..

The drug delivery system of U.S. Patent Application Publication No. 20110195123 is optionally associated with detection and / or activation devices, such devices, for example, optionally, but not by heat. By non-invasive and / or minimally invasive methods of activation and / or acceleration / deceleration, including heating and cooling, laser beams, and focused ultrasound and / or ultrasound including RF (radio frequency) methods or devices. , Activated during and / or after implantation of the device.

According to some embodiments of U.S. Patent Application Publication No. 20110195123, sites of local delivery include abnormally high proliferation and inhibition of apoptosis of cells, including tumors, activity and / or chronicity, including autoimmune disease states. Inflammation and infection, degeneration of tissues including muscle and nerve tissue, chronic pain, degenerative sites, fracture sites and other wound sites that promote tissue regeneration, and injured myocardium, smooth muscle, and striated muscle Can optionally include a target site characterized by.

The implantation site, or target site, of the composition is preferably characterized by having a radius, area, and / or volume sufficiently small for target local delivery. For example, the target site optionally has a diameter in the range of about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximum therapeutic effect. For example, the composition of the drug delivery system (optionally, together with the implantation device described above) is optionally and preferably implanted inside or near the tumor environment or a blood supply associated with the tumor environment. ..

For example, the composition (optionally with the device) is optionally implanted in or near the pancreas, prostate, breast, liver via the nipple, such as in the vascular system.

The target position is: 1. 2. Brain at degenerated sites such as Parkinson's disease or Alzheimer's disease in the basal ganglia, white matter, and gray matter; Spine in the case of amyotrophic lateral sclerosis (ALS); 3. Cervix to prevent HPV infection; 4. Active and chronically inflamed joints; 5. Dermis in case of psoriasis; 6. Sympathetic and sensory nerve sites for analgesic effect; 7. In bone graft; 8. Acute and chronic infection sites; 9. Intravaginal; 10. Inner ear-auditory system, inner ear labyrinth, vestibular system; 11. Intratracheal; 12. Intracardiac; coronary artery, epicardium; 13. Bladder; 14. Bile duct system; 15. Parenchymal tissue, including but not limited to kidney, liver and spleen; 16. Lymph nodes; 17. Salivary glands; 18. Gingiva; 19. Intra-articular (inside the joint); 20. Intraocular; 21. Brain tissue; 22. Ventricles; 23. Cavity, including the abdominal cavity (eg, for, but not limited to, ovarian cancer); 24. In the esophagus and 25. Arbitrarily selected from the group consisting within the rectum (simply, as a non-limiting example, optionally, any part of the body may be suitable for Loder implantation).

Optionally, the insertion of a system (eg, a device containing the composition) involves injecting material into the ECM of the target site and the vicinity of that site to local pH and / or temperature in and near the target site and such site. / Or related to influencing other biological factors that affect drug diffusion and / or pharmacokinetics in the ECM.

Optionally, according to some embodiments, the release of the agent may be associated with a detection instrument and / or an activator, such as a laser beam, irradiation, thermal heating and cooling, and focusing. By non-invasive and / or minimally invasive and / or other methods of activation and / or acceleration / deceleration, including ultrasound and / or ultrasound including RF (radio frequency) methods or devices, and chemical activators. Activated before and / or during and / or after insertion.

According to other embodiments of U.S. Patent Application Publication No. 20110195123, the drug is preferably for localized cancers in the breast, pancreas, brain, kidneys, bladder, lungs, and prostate, eg, as described below. Includes RNA. As exemplified by RNAi, if many drugs are applicable for encapsulation in a Loder and such drugs can be encapsulated in a Loder substrate, such as a matrix, they will be used in connection with the present invention. This system can be used and / or adapted for delivery of the CRISPR Cas system of the present invention.

As another example of a particular application, degenerative diseases of nerves and muscles are caused by aberrant gene expression. Local delivery of RNA can have therapeutic properties that prevent such aberrant gene expression. Topical delivery of anti-apoptotic, anti-inflammatory, and anti-denaturing agents, including small drugs and macromolecules, can also optionally be therapeutic. In such cases, the Loder is applied for long-term release by a dedicated device implanted at a constant rate and / or separately. All of this can be used and / or adapted to the CRISPR Cas system of the present invention.

As yet another example of a particular application, psychiatric disorders and cognitive deficits are treated with genetically modified agents (gene modifiers). Gene knockdown is a treatment option. Loders that deliver the agent locally to the central nervous system site are treatment options for psychiatric and cognitive disorders, including, but not limited to, psychiatric disorders, bipolar disorders, neurotic disorders, and behavioral disorders. .. Loder can also locally deliver drugs containing small drugs and macromolecules when implanted in specific parts of the brain. All of this can be used and / or adapted to the CRISPR Cas system of the present invention.

As another example of a particular application, silencing of congenital and / or adaptive immunomediators at a local site can prevent organ transplant rejection. By repelling immune cells activated against the transplanted organ, such as CD8, by local delivery of RNA and immunosuppressants in the transplanted organ and / or the Loder implanted at the implant site Local immunosuppression becomes possible. All of this can be used and / or adapted to the CRISPR Cas system of the present invention.

As another example of a particular application, angioplasty including VEGF and angiogenin is essential for neovascularization. Topical delivery of factors, peptides, peptide mimetics, or suppression of these repressors is an important mode of treatment; factors, peptides, macromolecules, and factors that stimulate repressor silencing and angiogenesis in the Loder. Topical delivery of small drugs has therapeutic effects on peripheral vascular disease, systemic vascular disease, and cardiovascular disease.

The method of insertion, eg, implantation, is optionally without any modification in such a method, or otherwise with any minor modification, optionally implantation and / or insertion and / or tissue of other types of tissue. May already be used for sampling. Such methods are optional, but not limited to, small radiation source therapy, biopsy, endoscopy with and / or without ultrasound, such as ERCP, stereotaxic method of insertion into brain tissue. Includes laparoscopy, including implantation of a laparoscopy into joints, abdominal organs, bladder walls, and body cavities.

The implantable device technology discussed herein can be used using the teachings herein, and thus with this disclosure and knowledge in the art, coding the CRISPR-Cas system or its constituents or constituents. The nucleic acid molecule provided or provided may be delivered by an implantable device.

Aerosol Delivery Subjects treated for lung disease may, for example, receive transpulmonary administration of a pharmaceutically effective amount of an aerosolized AAV vector system delivered intrabronchially under spontaneous breathing. As such, aerosolized delivery is generally preferred for AAV delivery. Adenovirus or AAV particles can be used for delivery. Suitable gene constructs, each operably linked to one or more regulatory sequences, can be cloned into the delivery vector. In this case, the following constructs are provided by way of example: Cbh or EF1a promoter for Cas (Cas9), U6 or H1 promoter for guide RNA): preferred configurations are CFTRΔ508 targeting guide, ΔF508 mutation repair template and codons. The optimized Cas9 enzyme is optionally used with one or more nuclear localization signals or sequences (NLS), such as two NLS. Constructs that do not contain NLS are also envisioned.

CRISPR enzyme mRNA and guide RNA
The CRISPR enzyme mRNA and guide RNA may also be delivered separately. The CRISPR enzyme mRNA may be delivered prior to the guide RNA to give the CRISPR enzyme time to express. The CRISPR enzyme mRNA may be administered 1-12 hours (preferably about 2-6 hours) prior to administration of the guide RNA.

Alternatively, the CRISPR enzyme mRNA and guide RNA may be administered together. Advantageously, a second booster dose of guide RNA may be administered 1-12 hours (preferably about 2-6 hours) after the initial administration of the CRISPR enzyme mRNA + guide RNA.

Additional administration of CRISPR enzyme mRNA and / or guide RNA may be useful in achieving the most efficient levels of genomic modification. In some embodiments, especially when a hereditary disease is targeted in a therapeutic method, the phenotypic change is preferably the result of genomic modification, preferably to modify or alter the phenotype here. A repair template is provided.

In some embodiments, potentially targeted diseases include those associated with disease-causing splice deficiency.

In some embodiments, cell targets include hematopoietic stem / progenitor cells (CD34 +); human T cells; and eyes (retinal cells) -eg, photoreceptive progenitor cells.

In some embodiments, the gene target is human β-globin-HBB, including for the treatment of sickle cell anemia, by stimulation of gene conversion (using a closely related HBD gene as an endogenous template); CD3 (T cells); and CEP920-retina (eye).

In the discussion herein regarding the association of a target with a mutation or disease state, such mutation or disease state includes, for example, hemophilia B, SCID, SCID-X1, ADA-SCID, hereditary tyrosineemia. Sickle erythrocyte anemia, β-salasemia, X-chain CGD, Viscott Oldrich syndrome, Funcony anemia, adrenoleukodystrophy (ALD), heterozygous white dystrophy (MLD), Asher syndrome, retinal pigment degeneration, label congenital Leber's Continental Amaurosis, cystic fibrosis, HIV / AIDS, HSV-1, HSV-2; or more generally, immunodeficiency disorders, hematological conditions, or genetic lysosome accumulation. obtain. The target may be associated with immunotherapy, such as cancer immunotherapy.

In some embodiments, delivery methods include cationic lipid-mediated "direct" delivery of enzyme-guide complexes (ribonuclear proteins) and electroporation of plasmid DNA.

The methods of the invention can further include delivery of a template such as a repair template, which may be dsODN or ssODN (see below). Delivery of the template may be simultaneous or separate with delivery of some or all of the CRISPR enzyme, guide, tracrmate or tracrRNA, and may be by the same or different delivery mechanisms. In some embodiments, the template is preferably delivered with the guide, tracr mate and / or tracr RNA, preferably with the CRISPR enzyme as well. One example may be an AAV vector, where the CRISPR enzyme is SaCas9 (including N580 mutation).

The method of the present invention is (a) a step of delivering a double-stranded oligodeoxynucleotide (dsODN) containing an overhang complementary to the overhang caused by the double-strand break to cells, wherein the dsODN is an object. Incorporating into the locus of the gene; or-(b) delivering a single-stranded oligodeoxynucleotide (ssODN) to cells, wherein the ssODN acts as a template for homology-dependent repair of the double-stranded break. Can be further included. The method of the present invention may be a method of preventing or treating a disease of an individual, and the disease is optionally caused by a defect in the locus of interest. The method of the present invention can be performed in vivo in an individual or ex vivo on cells taken from an individual, and the cells are optionally returned to the individual.

The enzymes according to the invention can be applied in an optimized functional CRISPR-Cas system useful for functional screening; in some embodiments, the present invention is of type V, more specifically the purpose of the cell. Provided is a non-naturally occurring or engineered composition comprising a Cas9 CRISPR guide RNA comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of, where the guide RNA comprises two or more adapter proteins ( It is modified by the insertion of one or more individual RNA sequences that bind to, for example, an aptamer), and each adapter protein is associated with one or more functional domains; or where the guide RNA is at least one non-coding. Modified to have a functional loop. In a detailed embodiment, the guide RNA is modified by inserting one or more individual RNA sequences into the 5'side of the direct repeat, within the direct repeat, or to the 3'side of the guide sequence. When there are two or more functional domains, they may be the same or different, eg, the same two or two different activators or repressors. In some embodiments, the invention comprises a guide RNA as discussed herein and a CRISPR enzyme that is a Cas9 enzyme [optionally the Cas9 enzyme contains at least one mutation, so that the Cas9 enzyme is at least one abrupt. Has less than 5% nuclease activity of the unmutated Cas9 enzyme, and optionally contains one or more containing at least one nuclear localization sequence] and is not naturally present or engineered. Also provided is a CRISPR-Cas complex composition. In some embodiments, the invention provides a Cas9 CRISPR guide RNA or Cas9 CRISPR-Cas complex discussed herein, including a non-naturally occurring or engineered composition comprising two or more adapter proteins. Where each protein associates with one or more functional domains, and the adapter protein binds to one or more individual RNA sequences inserted into the guide RNA. In a detailed embodiment, the guide RNA, in addition to or in place of it, still ensures binding of the Cas9 CRISPR complex, but prevents cleavage by the Cas9 enzyme (discussed elsewhere herein). As), it will be modified.

In some embodiments, the present invention comprises a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a target genomic locus of a cell, a Cas9 enzyme comprising at least one or more nuclear localized sequences. Provided is a non-naturally occurring or engineered composition containing, wherein the Cas9 enzyme comprises at least one mutation, so that the Cas9 enzyme has less than 5% nuclease activity of the Cas9 enzyme having at least one mutation. Having, the guide RNA is modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and is the adapter protein associated with one or more functional domains; or the gRNA Modified to have at least one non-coding functional loop, and the composition comprises two or more adapter proteins, each of which is associated with one or more functional domains. In some embodiments, the invention provides the compositions discussed herein, wherein the Cas9 enzyme is at least 97% or 100% nuclease when compared to the Cas9 enzyme without at least one mutation. Has reduced activity. In some embodiments, the invention provides the compositions discussed herein, where the Cas9 enzyme comprises two or more mutations. In some embodiments, the invention provides the compositions discussed herein, where the Cas9 enzyme is associated with one or more functional domains. In some embodiments, the invention provides the compositions discussed herein, wherein the two or more functional domains associated with the adapter protein are each heterologous functional domain. In some embodiments, the invention provides the compositions discussed herein, wherein the one or more functional domains associated with the Cas9 enzyme are each heterologous functional domain. In some embodiments, the invention provides the compositions discussed herein, wherein the adapter protein is a fusion protein comprising a functional domain, which optionally comprises the adapter protein and the functional domain. A linker is included in between, and the linker optionally includes a GlySer linker. In some embodiments, the invention provides the compositions discussed herein, wherein the gRNA is modified by the insertion of one or more individual RNA sequences that bind to two or more adapter proteins. Absent. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the adapter protein are transcriptional activation domains. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are transcriptional activation domains. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the adapter protein include VP64, p65, MyoD1, HSF1, RTA or SET7 / 9. It is a transcriptional activation domain that contains. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are transcriptional activation domains, VP64, p65, MyoD1, HSF1,. Includes RTA or SET7 / 9. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the adapter protein are transcriptional repressor domains. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are transcriptional repressor domains. In some embodiments, the invention provides the compositions discussed herein, wherein the transcriptional repressor domain is the KRAB domain. In some embodiments, the invention provides the compositions discussed herein, where the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or SID4X domain. In some embodiments, the invention provides the compositions discussed herein, wherein at least one of the one or more functional domains associated with the adapter protein is methylase activity, demethylase activity, transcriptional activation activity. , Transcription inhibitory activity, transcription release factor activity, histone modification activity, DNA integration activity, RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity. , Transcription release factor activity, histone modification activity, DNA integration activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or molecular switch activity or one or more activities including chemical inducibility or photoinducibility. In some embodiments, the invention provides the compositions discussed herein, where the DNA-cleaving activity is due to the Fok1 nuclease. In some embodiments, the invention provides the compositions discussed herein, wherein when one or more functional domains bind to a gRNA and a target, the functional domain can function with its attribution function. It is added to the Cas9 enzyme so that the functional domain is placed in a spatial arrangement; or optionally, one or more functional domains are added to the Cas9 enzyme via a linker, optionally a GlySer linker. In some embodiments, the invention provides the compositions discussed herein, wherein the gRNA has its attribution function after the gRNA binds to the adapter protein and further binds to the Cas9 enzyme and target. It is modified so that the functional domain is placed in a spatial arrangement that can function in. In some embodiments, the invention provides the compositions discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are added to the RuvC domain of Cas9. In some embodiments, the invention provides the compositions discussed herein, where the direct repeat of a guide RNA is modified by the insertion of one or more individual RNA sequences. In some embodiments, the invention provides the compositions discussed herein, wherein the insertion of one or more individual RNA sequences that bind to one or more adapter proteins is an aptamer sequence. In some embodiments, the invention provides the compositions discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for the same adapter protein. In some embodiments, the invention provides the compositions discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for different adapter proteins. In certain embodiments, the present invention provides the compositions discussed herein, wherein the adapter proteins are MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500. , KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, PRR1. Thus, in a detailed embodiment, the aptamer is selected from binding proteins that specifically bind any one of the adapter proteins listed above. In some embodiments, the invention provides the compositions discussed herein, where the cell is a eukaryotic cell. In some embodiments, the invention provides the compositions discussed herein, wherein the eukaryotic cell is a mammalian cell, plant cell or yeast cell, whereby the mammalian cell is optionally a mouse cell. .. In some embodiments, the invention provides the compositions discussed herein, wherein the mammalian cell is a human cell. In some embodiments, the invention provides the compositions discussed herein, where the first adapter protein associates with the p65 domain and the second adapter protein associates with the HSF1 domain. In some embodiments, the invention provides the compositions discussed herein, wherein the composition is at least three functional domains, at least one of which associates with a Cas9 enzyme, of which Includes a CRISPR-Cas complex having a functional domain in which at least two of the gRNAs are associated with gRNA.

In some embodiments, the invention provides the compositions discussed herein above, where there are two or more gRNAs, and these gRNAs target different sequences, thereby being used by the composition. When it is, there is multiplexing. In some embodiments, the invention provides a composition with two or more gRNAs modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins.

In some embodiments, the invention provides the compositions discussed herein, wherein there are one or more adapter proteins associated with one or more functional domains, and these are inserted into a guide RNA. It binds to one or more individual RNA sequences.

In some embodiments, the invention provides the compositions discussed herein, wherein the one or more target sequences are non-coding sequences or regulatory sequences. The regulatory sequence can be one or more promoter, enhancer or silencer sequences.

In some embodiments, the invention provides the compositions discussed herein, wherein the guide RNA is modified to have at least one non-coding functional loop; for example, at least one non-coding functional loop. Loops are inhibitory; for example, at least one non-coding functional loop contains an Alu.

In some embodiments, the invention provides a method of introducing a genomic locus event, which method administers or administers one or more of the compositions as discussed herein to the host. Includes expression in vivo. In some embodiments, the invention provides the methods discussed herein, wherein genomic locus events affect gene activation, gene inhibition, or cleavage at a locus.

In some embodiments, the invention provides the methods discussed herein, where the host is a eukaryotic cell. In some embodiments, the invention provides the methods discussed herein, wherein the host is a mammalian cell, optionally a mouse or plant cell or yeast cell. In some embodiments, the invention provides the methods discussed herein, wherein the host is a non-human eukaryote. In some embodiments, the present invention provides the methods discussed herein, wherein the non-human eukaryote is a non-human mammal. In some embodiments, the invention provides the methods discussed herein, wherein the non-human mammal is a mouse.

In certain embodiments, the present invention provides a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing or expressing the composition as discussed herein in the cell. In certain embodiments, the present invention provides the methods discussed herein, comprising delivering the composition or one or more nucleic acid molecules encoding it, wherein said one or more nucleic acid molecules. It is operably linked to one or more regulatory sequences and expressed in vivo. In some embodiments, the invention provides the methods discussed herein, where expression in vivo is mediated by lentivirus, adenovirus, or AAV.

In some embodiments, the invention provides a mammalian cell line of cells as discussed herein, where the cell line is optionally a human or mouse cell line. In some embodiments, the invention provides a transgenic mammalian model, optionally a mouse, wherein the model has been transformed with the compositions discussed herein or is a progeny of said transformant. Is.

In some embodiments, the invention provides one or more nucleic acid molecules encoding a guide RNA or Cas9 CRISPR-Cas complex or composition as discussed herein. In some embodiments, the present invention provides a vector comprising a nucleic acid molecule encoding a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at the genomic locus of interest in a cell, wherein the gRNA is direct. The repeat is modified by the insertion of one or more individual RNA sequences that bind to two or more adapter proteins, and is each adapter protein associated with one or more functional domains; or the gRNA is at least one. Modified to have one non-coding functional loop. In some embodiments, the invention relates to the gRNA discussed herein and the Cas9 enzyme [optionally the Cas9 enzyme comprises at least one mutation, so that the Cas9 enzyme does not have at least one mutation. A non-naturally occurring or engineered CRISPR-Cas complex composition that has less than 5% nuclease activity and optionally contains at least one containing at least one nuclear localization sequence]. Provided are one or more vectors containing one or more nucleic acid molecules encoding an article. In some embodiments, the vector is operably linked to a nucleic acid molecule encoding a guide RNA (gRNA) and / or a nucleic acid molecule encoding a Cas9 enzyme and / or an optional nuclear localization sequence. It may further comprise one or more regulatory elements that can operate in eukaryotic cells.

In one aspect, the invention provides a kit comprising one or more of the constituents described above. In some embodiments, the kit includes a vector system as described above and instructions for use of the kit.

In some embodiments, the invention provides a method of screening for gain of function (GOF) or loss of function (LOF) or a method of screening for non-coding RNA or potential regulatory regions (eg enhancers, repressors). , Cas9-containing or expressing cell lines as discussed herein or model cells as discussed herein, and compositions as discussed herein cell lines or model cells. The gRNA introduced into the cell contains either an activator or a repressor, and the gRNA introduced with respect to those cells contains an activator, or the gRNA introduced with respect to those cells re. It involves monitoring the GOF or LOF, respectively, with respect to which one includes the presser. The screening of the present invention is referred to as SAM screening.

In some embodiments, the present invention provides a Cas9 CRISPR Cas complex comprising a Cas9 enzyme and a guide RNA (gRNA), wherein the Cas9 enzyme has at least one mutation [hence the Cas9 enzyme has at least one mutation. Has less than 5% nuclease activity of Cas9 enzyme], and optionally contains at least one or more nuclear localization sequences; guide RNAs (gRNAs) are target sequences at the cell's desired genomic locus. Contains a guide sequence capable of hybridizing to; and the gRNA is modified by insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and the adapter proteins associate with two or more functional domains. Or the gRNA is modified to have at least one non-coding functional loop; or the Cas9 enzyme is associated with one or more functional domains and the gRNA binds to one or more adapter proteins. It is modified by the insertion of one or more individual RNA sequences, and the adapter protein is associated with more than one functional domain, or the gRNA is modified to have at least one non-coding functional loop. ..

In certain embodiments, the present invention provides a genome-wide library comprising multiple Cas9 guide RNAs (gRNAs), each containing a guide sequence capable of hybridizing to a target sequence at the genomic locus of interest in the cell, and thereby. The library can target multiple target sequences at multiple genomic loci in the eukaryotic cell population, where each gRNA binds to one or more or more than one adapter protein. It is modified by insertion of an individual RNA sequence, and is the adapter protein associated with one or more functional domains; or the gRNA is modified to have at least one non-coding functional loop. And when there are two or more functional domains, the functional domains may be the same or different, eg, the same two or two different activators or repressors. In some embodiments, the invention presents the gRNA of the invention and the Cas9 enzyme [optionally the Cas9 enzyme contains at least one mutation, so that the Cas9 enzyme is less than or equal to 5% of the Cas9 enzyme without at least one mutation. A library of non-naturally occurring or engineered CRISPR-Cas complex compositions that have nuclease activity and optionally include one or more containing at least one nuclear localization sequence. provide. In some embodiments, the invention comprises one or more gRNAs or one or more Cas9 CRISPR-Cas of the invention comprising a non-naturally occurring or engineered composition comprising one or more adapter proteins. It provides a complex, where each protein associates with one or more functional domains, and the adapter protein binds to one or more individual RNA sequences inserted into at least one loop of gRNA.

In some embodiments, the present invention comprises a Cas9 CRISPR guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a target genomic locus of a cell, a Cas9 enzyme comprising at least one or more nuclear localized sequences. Each provides a library of non-naturally occurring or engineered compositions, wherein the Cas9 enzyme contains at least one mutation, so that the Cas9 enzyme is 5% of the Cas9 enzyme that does not have at least one mutation. Having the following nuclease activity, at least one loop of gRNA is modified by the insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and the adapter proteins are one or more functional domains. The composition comprises one or more or two or more adapter proteins, each protein associates with one or more functional domains, and the gRNA is genomic wide containing multiple Cas9 guide RNAs (gRNAs). Includes libraries. In some embodiments, the invention provides a library as discussed herein, wherein the Cas9 enzyme is at least 97% or 100% nuclease when compared to the Cas9 enzyme without at least one mutation. Has reduced activity. In some embodiments, the invention provides a library as discussed herein, where the Cas9 enzyme comprises two or more mutations. In some embodiments, the invention provides a library as discussed herein, where the Cas9 enzyme comprises two or more mutations. In some embodiments, the invention provides a library as discussed herein, where the Cas9 enzyme is associated with one or more functional domains. In some embodiments, the invention provides a library as discussed herein, wherein the one or more functional domains associated with the adapter protein are heterologous functional domains. In some embodiments, the invention provides a library as discussed herein, wherein the one or more functional domains associated with the Cas9 enzyme are heterologous functional domains. In some embodiments, the invention provides a library as discussed herein, wherein the adapter protein is a fusion protein comprising a functional domain. In some embodiments, the invention provides a library as discussed herein, wherein the gRNA is an insertion of one or more individual RNA sequences that bind to one or more adapter proteins. Not modified by. In some embodiments, the invention provides a library as discussed herein, wherein the one or more functional domains associated with the adapter protein are transcriptional activation domains. In some embodiments, the invention provides a library as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are transcriptional activation domains. In some embodiments, the invention provides a library as discussed herein, wherein one or more functional domains associated with the adapter protein are transcriptional activity comprising VP64, p65, MyoD1 or HSF1. It is a conversion domain. In some embodiments, the invention provides a library as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are transcriptional activation domains, VP64, p65, MyoD1 or HSF1. including. In some embodiments, the invention provides a library as discussed herein, wherein the one or more functional domains associated with the adapter protein are transcriptional repressor domains. In some embodiments, the invention provides a library as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are transcriptional repressor domains. In some embodiments, the invention provides a library as discussed herein, where the transcriptional repressor domain is the KRAB domain. In some embodiments, the invention provides a library as discussed herein, wherein the transcriptional repressor domain is a SID domain or a SID4X domain. In some embodiments, the invention provides a library as discussed herein, wherein at least one of one or more functional domains associated with an adapter protein is methylase activity, demethylase activity. It has one or more activities including transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity. In some embodiments, the invention provides a library as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity. , Transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or molecular switch activity or one or more activities including chemical inducibility or photoinducibility. In some embodiments, the invention provides a library as discussed herein, where the DNA cleavage activity is Fok1 nuclease. In some embodiments, the invention provides a library as discussed herein, wherein when one or more functional domains bind to a gRNA and a target, the functional domain can function with its attribution function. It is added to the Cas9 enzyme so that the functional domain is placed in a spatial arrangement. In some embodiments, the invention provides a library as discussed herein, wherein the gRNA has its attribution function after the gRNA binds to the adapter protein and further binds to the Cas9 enzyme and target. The functional domain is modified to be placed in a spatial arrangement that can function in. In some embodiments, the invention provides a library as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are added to the N-terminus of the Cas9 enzyme. In some embodiments, the invention provides a library as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are RuvC of the FnCas9 protein or any ortholog corresponding to these domains. Is added to. In some embodiments, the invention provides a library as discussed herein, where direct repeats of gRNAs are modified by the insertion of one or more individual RNA sequences. In some embodiments, the invention provides a library as discussed herein, wherein the insertion of one or more individual RNA sequences that bind to one or more adapter proteins is an aptamer sequence. .. In some embodiments, the invention provides a library as discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for the same adapter protein. In some embodiments, the invention provides a library as discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for different adapter proteins. In some embodiments, the invention provides a library as discussed herein, wherein the adapter proteins are MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, Includes JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, PRR1. In some embodiments, the invention provides a library as discussed herein, wherein the cell population of cells is a eukaryotic cell population. In some embodiments, the invention provides a library as discussed herein, where eukaryotic cells are mammalian cells, plant cells or yeast cells. In some embodiments, the invention provides a library as discussed herein, wherein the mammalian cell is a human cell. In some embodiments, the invention provides a library as discussed herein, wherein the cell population is a population of embryonic stem (ES) cells. In some embodiments, the invention provides a library as discussed herein, where the target sequence at the genomic locus is a non-coding sequence. In some embodiments, the invention provides a library as discussed herein, where said targeting alters the gene function of one or more gene products; or is there a function acquisition with respect to gene function? Or is there a functional change in gene function; or is there a functional decline in gene function; or screening is for non-coding RNA or potential regulatory regions (eg enhancers, repressors). In some embodiments, the invention provides a library as discussed herein, where said targeting results in knockout of gene function. In some embodiments, the invention provides a library as discussed herein, in which more than about 100 sequences are targeted. In some embodiments, the invention provides a library as discussed herein, in which more than about 1000 sequences are targeted. In some embodiments, the invention provides a library as discussed herein, in which more than about 20,000 sequences are targeted. In some embodiments, the invention provides a library as discussed herein, where it targets the entire genome. In some embodiments, the invention provides a library as discussed herein, where it targets a panel of target sequences that focus on relevant or desirable pathways. In some embodiments, the invention provides a library as discussed herein, where the pathway is an immune pathway. In some embodiments, the invention provides a library as discussed herein, where the pathway is the cell division pathway. In some embodiments, the present invention provides a library as discussed herein, wherein changes in gene function are described in I.I. Cas9 protein, and II. Vectors for one or more vectors containing an engineered non-naturally occurring Cas9 CRISPR-Cas system containing one or more Cas9 guide RNAs [components I and II may be on the same or different vectors of the system] Steps to introduce the system into each cell in the cell population, step to integrate components I and II into each cell [The guide sequence targets a unique gene in each cell, and the Cas9 protein is operably linked to a regulatory element. The guide RNA containing the guide sequence, when transcribed, leads to sequence-specific binding of the Cas9 CRISPR-Cas system to the target sequence at the genomic locus of the unique gene], cleavage of the genomic locus by the Cas9 protein. Includes the step of inducing and identifying different mutations in multiple unique genes within each cell of the cell population, thereby generating a mutant cell library. In some embodiments, the invention provides a library as discussed herein, wherein one or more vectors are plasmid vectors. In some embodiments, the invention provides a library as discussed herein, where the regulatory element is an inducible promoter. In some embodiments, the invention provides a library as discussed herein, wherein the inducible promoter is a doxycycline inducible promoter. In some embodiments, the invention provides a library as discussed herein, where the step of identifying different mutations is by whole exome sequencing. In some embodiments, the invention provides a library as discussed herein, where mutations are realized in 100 or more unique genes. In some embodiments, the invention provides a library as discussed herein, where mutations are realized in over 1000 unique genes. In some embodiments, the invention provides a library as discussed herein, where mutations are realized in over 20,000 unique genes. In some embodiments, the invention provides a library as discussed herein, where mutations are realized throughout the genome. In some embodiments, the invention provides a library as discussed herein, wherein changes in gene function are realized in multiple unique genes that function in a particular physiological pathway or condition. In some embodiments, the invention provides a library as discussed herein, wherein the pathway or condition is an immune pathway or condition. In some embodiments, the invention provides a library as discussed herein, wherein the pathway or condition is a cell division pathway or condition. In some embodiments, the invention provides a library as discussed herein, where the first adapter protein associates with the p65 domain and the second adapter protein associates with the HSF1 domain. In some embodiments, the invention provides a library as discussed herein, wherein each Cas9 CRISPR-Cas complex is at least three functional domains, one of which is with the Cas9 enzyme. It has a functional domain that is associated and at least two of them are associated with gRNA. In some embodiments, the invention provides a library as discussed herein, where changes in gene function are knockout mutations.

In some embodiments, the present invention provides a method of functional screening of genomic genes in a cell pool, including administration or expression of a library containing multiple Cas9 CRISPR-Cas-based guide RNAs (gRNAs), exovivo or in vivo. Here the screening further involves the use of Cas9 enzymes and the CRISPR complex is modified to include a heterologous functional domain. In some embodiments, the invention provides a method for screening a genome, comprising administering the library to a host or expressing it in vivo in the host. In some embodiments, the invention provides a method as discussed herein, further comprising an activator administered or expressed in the host. In some embodiments, the invention provides the method as discussed herein, where the activator is added to the Cas9 enzyme. In some embodiments, the invention provides the method as discussed herein, where the activator is added to the N-terminus or C-terminus of the Cas9 enzyme. In some embodiments, the invention provides the method as discussed herein, where the activator is added to the Cas9 CRISPR gRNA direct repeat. In some embodiments, the invention provides a method as discussed herein, further comprising a repressor administered or expressed in the host. In some embodiments, the invention provides a method as discussed herein, wherein screening affects and detects gene activation, gene inhibition, or cleavage at a locus. including. In some embodiments, the invention provides the method as discussed herein, where the host is a eukaryotic cell. In some embodiments, the invention provides the method as discussed herein, where the host is a mammalian cell, yeast cell or plant cell. In some embodiments, the invention provides the method as discussed herein, where the host is a non-human eukaryote. In some embodiments, the invention provides the method as discussed herein, wherein the non-human eukaryote is a non-human mammal. In some embodiments, the invention provides the method as discussed herein, wherein the non-human mammal is a mouse. In some embodiments, the invention comprises the delivery of the Cas9 CRISPR-Cas complex or one or more components thereof or one or more nucleic acid molecules encoding them, as discussed herein. Where the one or more nucleic acid molecules are operably linked to one or more regulatory sequences and expressed in vivo. In some embodiments, the invention provides the method as discussed herein, where in vivo expression is mediated by lentivirus, adenovirus, or AAV. In some embodiments, the invention provides the method as discussed herein, where delivery is via particles, nanoparticles, lipids or cell permeable peptides (CPPs).

In some embodiments, the present invention provides a pair of Cas9 CRISPR-Cas complexes, each containing a Cas9 guide RNA (gRNA) containing a guide sequence capable of hybridizing to a target sequence at the target genomic locus of the cell. Here, the gRNA is modified by insertion of one or more individual RNA sequences that bind to one or more adapter proteins, and the adapter proteins associate with one or more functional domains, each of Cas9 CRISPR-Cas. The gRNA contains a functional domain having DNA cleaving activity. In some embodiments, the present invention provides a pair of Cas9 CRISPR-Cas complexes as discussed herein, where the DNA cleavage activity is due to Fok1 nuclease.

In the detailed embodiments of the methods and compositions herein, nucleotide sequences encoding the Cas9 protein codon-optimized for expression in eukaryotic cells are utilized. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell, yeast cell or plant cell. Alternatively, the eukaryotic cell is a plant cell. In a further embodiment of the invention, expression of the gene product is reduced.

In some embodiments of the methods and compositions provided herein, the Cas9 enzyme is an Acidaminococcus sp. BV3L6, a Lachnospiraceae bacterium MA2020 or a Francisella tularensis 1 Francisella. tularensis 1 Novicida) Cas9, which may include mutant Cas9 derived from these organisms. The enzyme may be Cas9 homolog or ortholog.

In one aspect, the invention provides a method of creating a model eukaryotic cell containing a gene whose expression has been modified. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method (a) introduces one or more of the vectors described above herein into eukaryotic cells, and (b) binds the CRISPR complex to a target polynucleotide. To create a model eukaryotic cell containing the modified locus, comprising the step of modifying the locus.

In one aspect, the invention provides a method of developing a biologically active agent that regulates cell signaling events associated with a disease gene. In some embodiments, the disease gene is any gene that is associated with an increased risk of developing or developing the disease. In some embodiments, the method involves (a) contacting the test compound with the model cell of any one of the embodiments described above; and (b) cell signaling associated with the mutation in the disease gene. The biologically active agent is developed that comprises the step of detecting a change in reading that is an indicator of decreased or increased events, thereby regulating the cell signaling event associated with the disease gene.

The present invention includes an optimized functional CRISPR-Cas Cas9 enzyme system, specifically combined with this modification guide, where the Cas9 enzyme is also associated with a functional domain. In particular, the Cas9 enzyme comprises one or more mutations that convert a functional domain exhibiting the desired function into a DNA binding protein that can be recruited, added, inserted or bound. In certain embodiments, the Cas9 enzyme comprises one or more mutations, and / or one or more mutations are in the RuvC1 domain of the Cas9 enzyme, or other mutations as discussed herein. Is. In some embodiments, the Cas9 enzyme has one or more mutations in the catalytic domain, where the guide sequence, when transcribed, leads to sequence-specific binding of the CRISPR complex to the target sequence, and The enzyme further comprises a functional domain. In some embodiments, mutations in E1006 relating to the FnCas9 protein are preferred.

The structural information provided herein allows the study of guide RNA interactions with the target DNA and Cas9 enzyme, and the guide RNA structure is optimized so that the functionality of the entire Cas9 CRISPR-Cas system is optimized. It will be possible to engineer or change. For example, the insertion of an adapter protein capable of binding RNA can prolong the loop of the guide RNA without colliding with the Cas9 protein. These adapter proteins can also recruit effector proteins or fusions that contain one or more functional domains.

In some preferred embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is a SID, or a concatemer of the SID (eg, SID4X). In some embodiments, the functional domain is an epigenetic modification domain, which provides an epigenetic modification enzyme. In some embodiments, the functional domain is the activation domain, which may be the P65 activation domain.

One aspect of the invention is that the above elements are included in a single composition or in individual compositions. These compositions can advantageously be applied to the host to elicit functional effects at the genomic level.

In general, guide RNAs are modified to provide specific binding sites (eg, aptamers) to which adapter proteins containing one or more functional domains bind (eg, via fusion proteins). The modified guide RNA is placed on the adapter protein in a spatial arrangement that favors binding of the adapter protein when the guide RNA forms a CRISPR complex (ie, the guide RNA and the Cas9 enzyme that binds to the target), enabling attribution function. The functional domain is modified to be positioned. For example, if the functional domain is a transcriptional activator (eg, VP64 or p65), the transcriptional activator is placed in a spatial arrangement in which it can affect the transcription of the target. Similarly, the transcription repressor will be advantageously positioned to influence the transcription of the target, and the nuclease (eg, Fok1) will be advantageously positioned to cleave or partially cleave the target. It will be.

Those skilled in the art allow the binding of the adapter + functional domain, but the adapter + functional domain cannot be properly located (eg, due to steric hindrance within the three-dimensional structure of the CRISPR complex). Will understand that it is an unintended modification. The modified guide RNA may be modified by introducing one or more individual RNA sequences into the 5'side of the direct repeat, within the direct repeat, or to the 3'side of the guide sequence.

As described herein, functional domains include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity. , And one or more domains from the group consisting of molecular switches (eg, photoinducible). In some cases it is advantageous to provide at least one more NLS. In some cases, it is advantageous to position the NLS at the N-terminus. When two or more functional domains are included, they may be the same or different.

Guide RNAs can be designed to contain multiple binding recognition sites (eg, aptamers) that are specific for the same or different adapter proteins. The guide RNA for the Cas9 enzyme is characterized by that it is typically 37-43 nucleotides and that it contains only one stem-loop. Guide RNAs can be designed to bind to the promoter region upstream of -1000 to +1 nucleic acids, preferably -200 nucleic acids, at the transcription initiation site (ie, TSS). Positioning in this way improves the functional domains that affect gene activation (eg, transcriptional activators) or gene inhibition (eg, transcriptional repressors). The modified guide RNA includes one or more target gene loci (eg, at least 1 guide RNA, at least 2 guide RNAs, at least 5 guide RNAs, at least 10 guide RNAs, at least 20 guide RNAs) contained in the composition. Guide RNA, at least 30 guide RNAs, at least 50 guide RNAs) may be one or more modified guide RNAs that target.

In addition, Cas9 enzymes with reduced nuclease activity were at least 70%, at least 80%, at least 90%, at least 95%, at least 97% when nuclease activity was inactivated (eg, when compared to wild-type enzymes. Or 100% nuclease inactivation; or, in other words, approximately 0% of the nuclease activity of the non-mutated or wild Cas9 enzyme, or nuclease activity of the non-mutated or wild Cas9 enzyme. Cas9 enzyme), which is about 3% or about 5% or less than about 10% of the most effective. This is possible by introducing a mutation into the RuvC nuclease domain of FnCas9 or its ortholog. For example, mutations in residues selected from the group consisting of D917A, E1006A, E1028A, D1227A, D1255A or N1257 as in FnCas9 are utilized, more preferably the positions D917A, E1006A, E1028A, D1227A, D1255A of FnCas9. , N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257 or one or more of the mutations selected from the group consisting of the corresponding orthologs. In a detailed embodiment, the mutation is D917A with E1006A in FnCas9.

Inactivated Cas9 enzymes include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switch (eg,). One or more functional domains are associated (eg, via a fusion protein), including one or more domains from the group consisting of (photoinducible), eg, as described herein for a modified guide RNA adapter protein. You may be doing it. Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. When Fok1 is provided, multiple Fok1 functional domains are provided to achieve a functional dimer, and guide RNAs are described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014), it is advantageous to be designed to provide appropriate spacing for functional use (Fok1). The adapter protein can utilize known linkers to add such functional domains. In some cases, it is advantageous to provide at least one additional NLS. In some cases, it is advantageous to position the NLS at the N-terminus. When two or more functional domains are included, they may be the same or different.

In general, the location of one or more functional domains on the inactivated Cas9 enzyme allows for the correct spatial placement for the functional domains to influence the target with the functional effect of attribution. For example, if the functional domain is a transcriptional activator (eg, VP64 or p65), the transcriptional activator is placed in a spatial arrangement in which it can affect the transcription of the target. Similarly, the transcription repressor is positioned to favorably affect the transcription of the target, and the nuclease (eg, Fok1) is positioned to favorably cleave or partially cleave the target. This may include positions other than the N-terminus / C-terminus of the Cas9 enzyme.

The adapter protein can be any protein that binds to an aptamer or recognition site introduced into the modified guide RNA, which affects the target with attribution function when the guide RNA is incorporated into the CRISPR complex. It is possible to properly locate one or more functional domains. As described in detail herein, this can be a coat protein, preferably a bacteriophage coat protein. Functional domains that associate with such adapter proteins (eg, in the form of fusion proteins) include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA. It may include one or more domains from the group consisting of cleavage activity, nucleic acid binding activity, and molecular switch (eg, photoinducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. If the functional domain is a transcription activator or transcription repressor, it is advantageous that at least NLS is preferably provided at the N-terminus. When two or more functional domains are included, they may be the same or different. The adapter protein can utilize known linkers to bind such functional domains. Such linkers can be used to associate DD with the CRISPR enzyme, or include DD in the CRISPR enzyme.

Thus, a modified guide RNA, an inactivated Cas9 enzyme (with or without a functional domain), and a binding protein having one or more functional domains are each individually included in the composition and individually or to the host. Can be administered in bulk. Alternatively, these components may be provided in a single composition for administration to a host. Administration to the host can be performed using viral vectors known to those of skill in the art for delivery to the host or described herein (eg, lentivirus vector, adenovirus vector, AAV vector). As described herein, the use of different selectable markers (eg, for lentiviral gRNA selection) and gRNA concentrations (eg, depending on whether multiple gRNAs are used) can improve efficacy. It can be advantageous to produce.

Based on this concept, several variations are appropriate for inducing genomic locus events, including DNA cleavage, gene activation, or gene deactivation. One of skill in the art can use the provided compositions to advantageously and specifically target single or multiple loci in the same or different functional domains to induce one or more genomic locus events. it can. The composition establishes cell lines and transgenic animals for screening in cell libraries and in vivo functional modeling (eg, identification of gene activation and function of lincRNA; functional acquisition modeling; loss of function modeling; optimization and screening purposes). Can be applied to a wide variety of methods for use of the compositions of the present invention).

The present invention includes the use of the compositions of the present invention for establishing and utilizing conditional or inducible CRISPR transgenic cells / animals (eg, Patent et al., Cell (2014), http: // PCT patents cited herein, such as dx. Doi.org / 10.1016 / j.cell. 2014.09.014, or WO 2014/093622 (PCT / US2013 / 074667). See publications (these are not considered to precede the invention or the present application). For example, the target cell conditionally or inducibly contained the Cas9 CRISPR enzyme (eg, in the form of a Cre-dependent construct) and / or the adapter protein conditionally or inducibly, and was introduced into the target cell. Upon expression of the vector, the vector is expressed to induce Cas9 enzyme expression and / or adapter expression in target cells, or to give rise to the conditions thereof. Inducible genomic events that are affected by the functional domain by applying the teachings and compositions of the invention along with known methods of creating CRISPR complexes are also aspects of the invention. A mere example of this is the production of CRISPR knock-in / conditional transgenic animals (eg, mice containing, for example, a Lux-termination-poly A-Lox (LSL) cassette), and subsequently as described herein ( One or more modification guide RNAs (eg, from -200 nucleotides to TSS of the target gene of interest for gene activation) (eg, a modification guide RNA having one or more aptamers recognized by a coat protein, eg MS2). , One or more adapter proteins as described herein (MS2-binding proteins linked to one or more VP64s) and means for inducing conditional animals (eg, Cre recombinases for inducing Cas9 expression). ) Is the delivery of one or more compositions. Alternatively, the adapter protein may be provided as a conditional or inducible element with a conditional or inducible Cas9 enzyme to provide a model that is effective for screening purposes, which is advantageous in a wide variety of applications. In contrast, it requires minimal design and administration of specific gRNA.

Deactivated / Inactivated Cas Protein When the Cas9 protein has nuclease activity, the Cas9 protein has reduced nuclease activity, eg, at least 70%, at least 80%, at least 90%, at least 95% when compared to wild-type enzymes. , May be modified to have at least 97%, or 100% nuclease inactivation; or in other words, the Cas9 enzyme is advantageously a non-mutated or wild Cas9 enzyme or CRISPR. About 0% of the nuclease activity of the enzyme, or about 3% or about 5% of the nuclease activity of a non-mutated or wild-type Cas9 enzyme, such as a non-mutated or wild-type Spyogenes Cas9 enzyme or CRISPR enzyme. % Or about 10% or less. This is possible by introducing mutations into the nuclease domain of Cas9 and its orthologs.

Inactivated Cas9 CRISPR enzymes include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switch ( One or more functional domains are associated (eg, via a fusion protein), including, for example, photoinducible), or essentially from them, or one or more domains from the group consisting of them. May be. Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. When Fok1 is provided, multiple Fok1 functional domains are provided to achieve a functional dimer, and sgRNAs are described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014), it is advantageous to be designed to provide appropriate spacing for functional use (Fok1). The adapter protein can utilize known linkers to add such functional domains. In some cases, it is advantageous to provide at least one additional NLS. In some cases, it is advantageous to position the NLS at the N-terminus. When two or more functional domains are included, they may be the same or different.

In general, the location of one or more functional domains on the inactivated Cas9 enzyme allows for the correct spatial placement for the functional domains to influence the target with the functional effect of attribution. For example, if the functional domain is a transcriptional activator (eg, VP64 or p65), the transcriptional activator is placed in a spatial arrangement in which it can affect the transcription of the target. Similarly, the transcription repressor is positioned to favorably affect the transcription of the target, and the nuclease (eg, Fok1) is positioned to favorably cleave or partially cleave the target. This may include positions other than the N-terminus / C-terminus of the CRISPR enzyme.

In certain embodiments, Cas9 may contain one or more mutations (and thus one or more nucleic acid molecules encoding it may have one or more mutations. Mutations are artificially introduced. Mutations may include, but are not limited to, one or more mutations in the catalytic domain. Examples of catalytic domains associated with Cas9 enzymes include, but are not limited to, RuvC I, RuvC II. , RuvC III and HNH domains.

In certain embodiments, Cas9 may contain one or more mutations. The mutation may be an artificially introduced mutation and may include, but is not limited to, one or more mutations in the catalytic domain, eg, nickase. Examples of catalytic domains associated with the Cas enzyme include, but are not limited to, RuvC I, RuvC II, RuvC III, and HNH domains.

In certain embodiments, Cas9 can be used as a generic nucleic acid binding protein fused to or operably linked to a functional domain. Exemplary functional domains include, but are not limited to, translation initiation factors, translation activators, translational suppressors, nucleases, and more specifically ribonucleases, spliceosomes, beads, photoinducible / regulatory domains or chemical-inducible / Controllable domains can be mentioned.

In some embodiments, the unmodified nucleic acid targeting effector protein may have cleavage activity. In some embodiments, the RNA targeting effector protein is one or both nucleic acids (DNA or) in or near the location of the target sequence, such as within the target sequence and / or in the complement of the target sequence or in a sequence associated with the target sequence. RNA) can lead to strand breaks. In some embodiments, the nucleic acid targeting Cas9 protein is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50 from the first or last nucleotide of the target sequence. , 100, 200, 500, or more base pairs, which can lead to cleavage of one or both DNA or RNA strands. In some embodiments, the cut is blunt, i.e. can give rise to blunt ends. In some embodiments, the cut is adherent, i.e. can give rise to adherent ends. In some embodiments, the cleavage may be an adherent cleavage with a 5'overhang, eg, a 5'overhang of 1-5 nucleotides. In some embodiments, the cleavage may be an adherent cleavage with a 3'overhang, eg, a 3'overhang of 1-5 nucleotides. In some embodiments, the vector encodes a nucleic acid targeting Cas protein that may be mutated compared to the corresponding wild enzyme, so that the mutant nucleic acid targeting Cas protein is a target poly containing a target sequence. It lacks the ability to cleave one or both DNA or RNA strands of a nucleotide. As a further example, mutating two or more catalytic domains of Cas (RuvC I, RuvC II, and RuvC III or HNH domains) results in a mutant Cas that lacks substantially all RNA-cleaving activity. May be done. As described herein, the corresponding catalytic domain of the Cas9 effector protein is also mutated to produce mutant Cas9 that lacks all DNA cleavage activity or has substantially reduced DNA cleavage activity. Can be. In some embodiments, the nucleic acid targeting effector protein has an RNA-cleaving activity of the mutant enzyme of about 25%, 10%, 5%, 1%, 0.1% of the nucleic acid-cleaving activity of a non-mutant enzyme. When less than 0.01% or less, it can be considered to lack virtually all RNA cleavage activity; one example is zero mutant nucleic acid cleavage activity when compared to non-mutant form. Or it may be negligible. Effector proteins can be identified by reference to common enzyme classes that share homology with the largest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the effector protein is a type II protein such as Cas9. Derived means that Applicants are generally based on wild-type enzymes in the sense that the derived enzymes have a high degree of sequence homology with wild-type enzymes, but as is known in the art or herein. It means that it has been mutated (modified) in some way as described in the book.

Again, the terms Cas and CRISPR enzymes and CRISPR proteins and Cas proteins are used broadly synonymously and, unless otherwise apparent, such as by specifically referring to Cas9, at any reference point herein. By analogy, it will be understood to refer to the novel CRISPR effector proteins further described in this application. As mentioned above, many of the residue numbering used herein refers to effector proteins from the type II CRISPR locus. However, it will be understood that the present invention includes more effector proteins from other microbial species.

List of organisms as possible sources of Cas protein The Cas proteins include Streptococcus, Campylobacter, Nitratifragtor, Staphylococcus, and Pulvibaccus. , Rosebria, Neisseria, Gluconacetobacter, Azospiryllum, Sphaerochaeta, Sphaerochaeta, Lactobacillus, Lactobacillus, Lactobacillus Cas proteins from organisms of the genus, including (Corynebacter), may be included.

Cas9 proteins include Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaccus, Parvibaccus, Parvibaccus, , Gluconacetobacter, Azospiryllum, Sphaerochaeta, Lactobacillus, Eubacterium, a protein from the genus Eubacterium, or a protein from the genus Corinebacter. Can be included.

Preferred examples include Streptococcus pyogenes (Spyogenes) and Staphylococcus aureus (Saureus).

In certain embodiments, the Cas9 protein is, but is not limited to, the genus Corynebacter, the genus Suterella, the genus Legionella, the genus Treponema, the genus Fifilactor, the genus Eubacterium ( Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavivola, Flavilovola, Flavilovera (Azospirillum), Gluconacetobacter, Neisseria, Rosebulia, Parvibaculum, Staphylococcus, Staphylococcus, Staphylococcus, Staphylococcus ) And the orthologs of the genera including the genus Campylobacter. Species of organisms of this genus may be as discussed elsewhere herein.

Some methods for identifying the orthologs of CRISPR-Cas-based enzymes may include the step of identifying the tracr sequence in the genome of interest. Identification of the tracr sequence may involve the following steps: The step of searching the database for direct repeat or tracr mate sequences to identify the CRISPR region containing the CRISPR enzyme. A step of searching for homologous sequences in the CRISPR region flanking the CRISPR enzyme in both the sense and antisense directions. Steps to examine the transfer terminator and secondary structure. A step of identifying any sequence that is not a direct repeat or tracr mate sequence but has greater than 50% identity with a direct repeat or tracr mate sequence as a potential tracr sequence. The step of extracting a potential tracr sequence and analyzing it for the transcription termination sequence associated with it.

It will be appreciated that any of the functionality described herein can be engineered to be a CRISPR enzyme from other orthologs, including chimeric enzymes containing fragments from multiple orthologs. Examples of such orthologs are described elsewhere herein. Thus, the chimeric enzymes are, but are not limited to, Corynebacter, Suterella, Legionella, Treponema, Flavobacterium, Flavobacterium, Eubacterium. Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Flavobacterium, Flavobacterium, Flavobacterium, Flavobacterium, Flavobacterium , Gluconacetobacter, Neisseria, Rosebria, Parvibaculum, Staphylococcus, Staphylococcus, Nitrachiflacta It may contain fragments of the CRISPR enzyme ortholog of organisms, including the genus Campylobacter. The chimeric enzyme can include a first fragment and a second fragment, which can be fragments of the CRISPR enzyme ortholog of an organism of the genus or species listed herein. Advantageously, the fragment is derived from a different species of CRISPR enzyme ortholog.

It may be important to control the concentration of CRISPR enzyme mRNA and guide RNA delivered to minimize toxicity and off-target effects. Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing various concentrations in cell or animal models and analyzing the degree of modification at potential off-target genomic loci using deep sequencing. For example, for a guide sequence targeting the 5'-GAGTCCGAGCAGAGAGAAGAA-3'of the EMX1 gene in the human genome, using deep sequencing, the following two off-target loci, 1: 5'-GAGTCCTAGCAGGAGAGAAGAA-3' and 2: 5 The modification level in'-GAGTCTAAGCAGAAGAAGAA-3'can be evaluated. Concentrations that result in the highest levels of on-target modification while minimizing off-target modification levels should be selected for in vivo delivery.

Delivery: DNA / RNA or DNA / DNA or RNA / RNA or protein / RNA options In some embodiments, the components of the CRISPR system are in various forms, such as DNA / RNA or RNA / RNA combinations or protein RNA. Can be delivered at. For example, Cas9 may be delivered as a DNA-encoding polynucleotide or RNA-encoding polynucleotide, or as a protein. The guide may be delivered as a DNA coding polynucleotide or RNA. Any possible combination is envisioned, including mixed delivery.

In some embodiments, all of such combinations (DNA / RNA or DNA / DNA or RNA / RNA or protein / RNA).

In some embodiments, if Cas9 is delivered in protein form, it can be pre-assembled with one or more guides.

Delivery: nanoclew
In addition, the CRISPR system is described, for example, by Sun W et al, "Cocoon-like self-degradable DNA nanoclew for anticancer drug delivery". , JAm Chem Soc. 2014 Oct 22; 136 (42): 14722-5. doi: 10.1021 / ja508824. EPUB 2014 Oct 13. Or Sun W et al, "Self-Assembled DNA Nanoclews for the Effective CRISPR-Cas9 for Genome for Efficient Delivery of CRISPR-Cas9 for Genome Editing". , Angew Chem Int Ed Engl. 2015 Oct 5; 54 (41): 12029-33. doi: 10.10012 / anie. 201506030. It can be delivered using nanoclaws as described in EPub 2015 Aug 27.

Delivery-GalNAc
CRISPR complex components can be delivered by conjugating or associating with a transport moiety (eg, US Pat. No. 8,106,022; (Incorporated in the specification)). For example, a nucleic acid delivery strategy can be used to improve delivery of guide RNA, or messenger RNA or coding DNA that encodes a CRISPR complex component, including a CRISPR protein. For example, by incorporating modified RNA nucleotides into RNA, stability can be improved, immune stimulation can be reduced, and / or specificity can be improved (Deleavey, Glen F. et al., 2012, Chemistry & Biology, Volume). 19, Issue 8,937-954; Zalipsky, 1995, Advanced Drug Delivery Review 16: 157-182; See Chemistry and Veronese, 2003, Advanced Drug Delivery 77) 126: 12riv. GRNAs for more efficient delivery, such as reversible charge-neutralizing phosphate triester skeleton modifications that can be adapted to gRNA modifications to make them more hydrophobic and non-anionic and thereby improve cell invasion. Various constructs that can be used to modify nucleic acids such as, are described (Made BR et al., 2014, Nature Biotechnology 32, 1256-1261). In a further alternative embodiment, the RNA motif selected may be useful in mediating cell transfection (Magalhaes M., et al., Molecular Therapy (2012); 2013, 616-624). .. Similarly, aptamers can be adapted for delivery of CRISPR complex components, eg, by adding aptamers to gRNA (Tan W. et al., 2011, Trends in Biotechnology, December 2011, Vol. 29, No. 12). ).

In some embodiments, conjugation of triantennary N-acetylgalactosamine (GalNAc) to oligonucleotide components can be used to improve delivery, eg, delivery to selected cell types, eg, hepatocytes (. WO 2014118272 (see herein by reference); Nir, JK et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961). This can be considered sugar-based particles, and further details regarding other particle delivery systems and / or formulations are provided herein under the corresponding headings. GalNAc can therefore be considered as a particle in the sense of the other particles described herein, and general use and other considerations, such as delivery of said particles, apply to GalNAc particles as well. For example, using a phase conjugation strategy, a triantennary GalNAc cluster (molecular weight about 2000) activated as a PFP (pentafluorophenyl) ester was converted to a 5'-hexylamino modified oligonucleotide (5'-HA ASO, molecular weight about 8000 Da). It can be added to Φstergaard et al., Bioconjugate Chem., 2015, 26 (8), pp 1451-1455). Similarly, poly (acrylate) polymers are described for in vivo nucleic acid delivery (see WO 2013158141 (referred to herein by reference)). In a further alternative embodiment, premixes of CRISPR nanoparticles (or protein complexes) with naturally occurring serum proteins can be used to improve delivery (Akinc A et al, 2010, Molecular Therapy vol. 18 no. 7, 1357-1364).

Screening techniques are available to identify delivery enhancers, for example by screening chemical libraries (Gilleron J. et al., 2015, Nucl. Acids Res. 43 (16): 7984-8001). Techniques for assessing the efficiency of delivery vehicles such as lipid nanoparticles have also been described, which can be used to identify effective delivery vehicles for CRISPR components (Sahay G. et al., 2013, Nature Biotechnology). See 31,653-658).

In some embodiments, the addition of a functional peptide to the protein, such as a peptide that alters the hydrophobicity of the protein, may facilitate delivery of protein CRISPR components, eg, to improve functionality in vivo. Similarly, the CRISPR complex component protein can be modified to facilitate subsequent chemical reactions. For example, amino acids with groups that cause click chemistry may be added to the protein (Nickic I. et al., 2015, Nature Protocols 10, 780-791). In this type of embodiment, click chemical groups are then used to provide a wide variety of alternatives such as poly (ethylene glycol) for stability, cell permeable peptides, RNA aptamers, lipids, or carbohydrates such as GalNAc. Structures can be added. In a further alternative example, the CRISPR complex component protein is used to adapt the protein to cell invasion (see Svensen et al., 2012, Trends in Physical Sciences, Vol. 33, No. 4), eg, protein. Can be modified by adding a cell-permeable peptide to (Kaufman, W. Berkeley et al., 2015, Trends in Biochemical Sciences, Volume 40, CRISPR 12, 749-764; Korean and Protein, 2012, Tren. Vol.18, No.7). In a further alternative embodiment, the patient or subject may be pretreated with a compound or formulation that facilitates subsequent delivery of the CRISPR complex component.

Inducible system In some embodiments, the CRISPR enzyme can form a component of the inducible system. The inducible nature of this system may allow for gene editing or spatiotemporal control of gene expression using energy forms. The form of energy includes, but is not limited to, electromagnetic radiation, sound energy, chemical energy, and thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule 2-hybrid transcriptional activation systems (FKBP, ABA, etc.), or photoinducible systems (phytochrome, LOV domain, or crypto). Chromium) is included. In one embodiment, the CRISPR enzyme can be part of a photo-induced transcriptional effector (LITE) that induces sequence-specific changes in transcriptional activity. Light components can include CRISPR enzymes, photoresponsive cytochrome heterodimers (eg from Arabidopsis thaliana), and transcriptional activation / repressor domains. Further examples of inducible DNA-binding proteins and their uses are described in US Provisional Patent Application No. 61 / 736,465, US Provisional Patent Application No. 61 / 721,283 and WO 2014/018423. Provided in a pamphlet (incorporated by reference in its entirety by this specification).

Self-inactivation system After all copies of the gene in the intracellular genome have been edited, CRISPR / Cas9 expression does not need to continue in the cell anymore. In fact, sustained expression may be undesirable, such as when off-target effects occur at unintended genomic sites. Therefore, timed expression can be useful. Inducible expression provides one approach, but in addition, Applicants have engineered a self-inactivating CRISPR-Cas9 system that relies on the use of non-coding guide target sequences within the CRISPR vector itself. Thus, after initiation of expression, the CRISPR system can result in its own disruption, but before the disruption is complete, it may have time to edit the genomic copy of the target gene (normal point mutations in diploid cells). So, this requires at most two edits). Simply, the self-inactivating CRISPR-Cas system targets the coding sequence of the CRISPR enzyme itself, or one or more non-coding guide targeting sequences complementary to the unique sequence present in one or more of the following: Includes additional RNA (ie, guide RNA) that targets
(A) In a promoter that drives the expression of non-coding RNA elements,
(B) In the promoter that drives the expression of the Cas9 gene,
(C) Within 100 bp from the ATG translation start codon in the Cas9 coding sequence,
(D) Within the reverse terminal repeat sequence (iTR) of the viral delivery vector, eg, in the AAV genome.

In addition, the RNA can be delivered in a vector encoding the CRISPR complex, eg, a separate vector or the same vector. When provided by a separate vector, CRISPR RNA that targets Cas expression can be administered sequentially or simultaneously. When administered sequentially, the CRISPR RNA that targets Cas expression should be delivered, for example, after the CRISPR RNA intended for gene editing or genetic engineering. This period may be several minutes (eg, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes). This period may be several hours (eg, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours). This period may be several days (eg, 2 days, 3 days, 4 days, 7 days). This period may be several weeks (eg, 2 weeks, 3 weeks, 4 weeks). This period may be several months (eg, 2 months, 4 months, 8 months, 12 months). This period may be several years (2 years, 3 years, 4 years). In this way, the Cas enzyme associates with a first gRNA / chiRNA capable of hybridizing to a first target, such as one or more genomic loci of interest, and the desired one or more of the CRISPR-Cas system. Responsible for multiple functions (eg, genetic engineering); and subsequently the Cas enzyme can associate with a second gRNA / chiRNA capable of hybridizing to a sequence containing at least a portion of the Cas or CRISPR cassette. If the sequence encoding Cas protein expression is the target of a gRNA / chiRNA, the enzyme is blocked and the system self-inactivates. Similarly, as described herein, CRISPR RNA that targets Cas expression applied, for example, via liposomes, lipofections, particles, microvesicles, may be administered sequentially or simultaneously. Similarly, self-inactivation can be used to inactivate one or more guide RNAs used to target one or more targets.

In some embodiments, a hybridizable single gRNA is provided in the sequence downstream of the CRISPR enzyme initiation codon, which results in loss of CRISPR enzyme expression after some time. In some embodiments, one or more gRNAs that are hybridizable to one or more coding or non-coding regions of the polynucleotide encoding the CRISPR-Cas system are provided, thereby after some time, CRISPR-. One or more of the Cas systems, or in some cases all, are inactivated. In some aspects of this system, and without limitation by theory, cells can contain multiple CRISPR-Cas complexes, where a first subset of the CRISPR complex seeks to edit 1 At least one second that contains a first chiRNA capable of targeting one or more genomic loci and a second subset of the CRISPR complex can target a polynucleotide encoding the CRISPR-Cas system. Contains chiRNA, where the first subset of the CRISPR-Cas complex mediates the editing of one or more target genomic loci, and the second subset of the CRISPR-Cas complex ultimately mediated the CRISPR-Cas system. Inactivates, which inactivates further CRISPR-Cas expression in the cell.

Accordingly, the present invention provides a CRISPR-Cas system comprising one or more vectors for delivery to eukaryotic cells, wherein the one or more vectors are (i) CRISPR enzymes; (ii) intracellular. A first guide RNA that can hybridize to a target sequence; (iii) a second guide RNA that can hybridize to one or more target sequences in a vector encoding a CRISPR enzyme; (iv) at least one tracr mate sequence And (v) encode at least one tracr sequence, the first and second complexes can use the same tracr and tracr mate, thus differing only in the guide sequence, where expressed intracellularly. : The first guide RNA leads to sequence-specific binding of the first CRISPR complex to the intracellular target sequence; the second guide RNA is the second to the target sequence in the vector encoding the CRISPR enzyme. Derivation of sequence-specific binding of the CRISPR complex; the CRISPR complex comprises (a) a tracr mate sequence hybridized to a tracr sequence and (b) a CRISPR enzyme bound to a guide RNA, thus the guide RNA to its target sequence. Can be hybridized; and the second CRISPR complex inactivates the CRISPR-Cas system, preventing continued expression of the CRISPR enzyme by cells.

Further features such as one or more vectors, encoded enzymes, guide sequences, etc. are disclosed elsewhere herein. For example, one or both of the guide sequences may be part of a chiRNA sequence that provides guide, tracr mate and tracr sequences within a single RNA, so the system is (i) CRISPR enzyme; (ii). ) A sequence capable of hybridizing to a first target sequence in a cell, a first tracr mate sequence, and a first chiRNA containing a first tracr sequence; (iii) CRISPR enzyme, a second tracr mate sequence, and A second guide RNA that can be hybridized to the vector encoding the second tracr sequence can be encoded. Similarly, the enzyme can include one or more NLS and the like.

Various coding sequences (CRISPR enzyme, guide RNA, tracr and tracr mate) may be included in a single vector or in multiple vectors. For example, encoding an enzyme on one vector and various RNA sequences on another vector, or encoding an enzyme on one vector and one chiRNA, and the rest of the chiRNA on another vector, or any Other sorts are possible. In general, a system using a total of one or two different vectors is preferred.

When multiple vectors are used, it is possible to deliver them in disproportionate numbers, and ideally in excess of the vector encoding the first guide RNA compared to the second guide RNA. This helps delay the final inactivation of the CRISPR system until point Z, when genome editing is given the opportunity to occur.

The first guide RNA can target any target sequence of interest in the genome, as described elsewhere herein. The second guide RNA targets the sequence within the vector encoding the CRISPR Cas9 enzyme, thereby inactivating the expression of the enzyme from that vector. Therefore, the target sequence in the vector must be capable of inactivating expression. Suitable target sequences are, for example, near or within the translation initiation codon of the Cas9 coding sequence, within the non-coding sequence, within the promoter driving the expression of the non-coding RNA element, within the range of the promoter driving the expression of the Cas9 gene. , Within 100 bp from the ATG translation start codon in the Cas9 coding sequence and / or within the range of the reverse end repeats (iTR) of the virus delivery vector, eg, in the AAV genome. Double-strand breaks near this region can cause a frameshift of the Cas9 coding sequence, which can lead to loss of protein expression. An alternative target sequence for the "self-inactivating" guide RNA is intended to edit / inactivate the regulatory regions / sequences required for CRISPR-Cas9 system expression or vector stability. obtain. For example, if the promoter of the Cas9 coding sequence is disrupted, transcription can be inhibited or prevented. Similarly, if the vector contains sequences for replication, maintenance or stability, it is possible to target them. For example, a useful target sequence in an AAV vector is in the iTR. Other sequences useful for targeting can be promoter sequences, polyadenylation sites, and the like.

Furthermore, when the guide RNA is expressed in array format, the "self-inactivating" guide RNA, which targets both promoters at the same time, results in the excision of intervening nucleotides from within the CRISPR-Cas expression construct, effectively Leads to complete inactivation. Similarly, interlinging nucleotides can be excised when the guide RNA targets both ITRs, or when two or more other CRISPR-Cas components are targeted simultaneously. Self-inactivation as described herein is generally applicable with the CRISPR-Cas9 system to provide regulation of CRISPR-Cas9. For example, self-inactivation as described herein may be applied to mutations as described herein, such as CRISPR repair of extended disorders. As a result of this self-inactivation, CRISPR repair is only a transient activity.

The 5'end of a "self-inactivating" guide RNA (eg 1-10 nucleotides, preferably 1-5 nucleotides) as a means of ensuring that the target genomic locus is edited before CRISPR-Cas9 is arrested The addition of non-targeting nucleotides to the can be used to delay its processing and / or alter its efficiency.

In one embodiment of the self-inactivating AAV-CRISPR-Cas9 system, one or more sgRNA targeting genomic sequences of interest (eg 1-2, 1-5, 1-10, 1-15, 1-20, 1-30). A plasmid co-expressing the seeds targets the SpCas9 sequence at or near the engineered ATG initiation site (eg, within 5 nucleotides, within 15 nucleotides, within 30 nucleotides, within 50 nucleotides, within 100 nucleotides). It can be made using "activated" sgRNA. Regulatory sequences in the U6 promoter region can also be targeted with sgRNA. U6 drive sgRNAs can be designed in an array format that allows the simultaneous release of multiple sgRNA sequences. When first delivered to the target tissue / cell, sgRNA (leaving the cell) begins to accumulate while elevated Cas9 levels in the nucleus. Cas9 forms a complex with all sgRNAs and mediates genome editing and self-inactivation of the CRISPR-Cas9 plasmid.

One aspect of the self-inactivating CRISPR-Cas9 system is alone or in a tandem array format with 1 to up to 4 or more different guide sequences; for example up to about 20 or about 30 guide sequences. Is the expression of. Different targets can be targeted for each individual self-inactivating guide sequence. It can be processed, for example, from one chimeric pol3 transcript. A Pol3 promoter such as the U6 or H1 promoter can be used. Pol2 promoters such as those referred to throughout herein. The reverse terminal repeat (iTR) sequence can be flanked by one or more sgRNA-Pol2 promoters-Cas9.

One aspect of the chimeric tandem array transcript is that one or more guides edit one or more targets while one or more self-inactivating guides inactivate the CRISPR / Cas9 system. .. Thus, for example, the described CRISPR-Cas9 system for repairing swelling disorders may be directly combined with the self-activating CRISPR-Cas9 system described herein. Such a system may have, for example, two guides directed to the target region for repair and at least a third guide directed to self-inactivation of CRISPR-Cas9. International Publication No. 2015/089351 Pamphlet published on December 12, 2014, "Compositions And Methods Of Use Of Crispr-Cas Systems InNucleotide Repe. The application PCT / US2014 / 069897, entitled "."

Kit In one aspect, the invention provides a kit comprising any one or more of the elements disclosed in the methods and compositions described above. The elements may be provided individually or in combination, and may be provided in any suitable container, such as a vial, bottle, or tube. In some embodiments, the kit comprises instructions in one or more languages, eg, two or more languages.

In some embodiments, the kit comprises one or more reagents used in methods that utilize one or more of the elements described herein. Reagents can be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. Reagents are provided in a form that can be used in a particular assay, or in a form that requires the addition of one or more other components prior to use (eg, concentrated or lyophilized forms). May be good. The buffer can be, but is not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and any buffer including any combination thereof. It may be there. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to the guide sequences inserted into the vector to operably link the guide sequences and regulatory elements. In some embodiments, the kit comprises a homologous recombination template polynucleotide. In some embodiments, the kit comprises one or more of the vectors and / or one or more polynucleotides described herein. The kit is advantageously capable of providing all the elements of the system of the invention.

Nucleic Acid Targeting System and Method The term "nucleic acid targeting system" (nucleic acid is DNA or RNA, and in some embodiments may also refer to a DNA-RNA hybrid or derivative thereof) collectively refers to DNA or RNA targeting CRISPR-related ( "Cas") refers to transcripts and other elements involved in the expression of a gene or the induction of its activity, including, but not all, sequences encoding DNA or RNA targeting Cas proteins and CRISPR RNA (crRNA) sequences. Can include a DNA or RNA targeting guide RNA containing a trans-activated CRISPR / Cas system RNA (tracrRNA) sequence (in some systems), or other sequences and transcripts from the DNA or RNA targeting CRISPR locus. In general, RNA targeting systems are characterized by elements that facilitate the formation of DNA or RNA targeting complexes at the site of the target DNA or RNA sequence. In the context of the formation of a DNA or RNA targeting complex, "target sequence" refers to a DNA or RNA sequence designed so that the DNA or RNA targeting guide RNA is complementary to it, where the target sequence and RNA targeting. Hybridization with guide RNA facilitates the formation of RNA targeting complexes. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

In certain aspects of the invention, the novel DNA targeting system, also referred to as the CRISPR / Cas or CRISPR-Cas DNA targeting system of the present application, does not require the creation of customized proteins to target specific DNA sequences. Rather, a single effector protein or enzyme can be programmed to recognize a specific DNA target by an RNA molecule, in other words, the RNA molecule can be used to recruit an enzyme to a specific DNA target. Based on the Cas9 protein produced. Aspects of the present invention particularly relate to the DNA targeting RNA guided SpCas9 CRISPR system.

In one aspect, the invention provides a method of using one or more elements of a nucleic acid targeting system. The nucleic acid targeting complex of the present invention provides an effective means of modifying a target DNA or RNA (single or double strand, linear or supercoiled). The nucleic acid targeting complex of the present invention has a wide range of usefulness, including modification of target DNA or RNA in a large number of cell types (eg, deletion, insertion, translocation, inactivation, activation). Thus, the nucleic acid targeting complex of the present invention has wide applicability in, for example, gene therapy, drug screening, disease diagnosis, and prognosis determination. An exemplary nucleic acid targeting complex comprises a DNA or RNA targeting effector protein that hybridizes with a guide RNA that hybridizes to a target sequence within the target locus of interest.

Nucleic acid targeting systems, vector systems, vectors and compositions described herein include various nucleic acid targeting applications, alterations or alterations in the synthesis of gene products such as proteins, nucleic acid cleavage, nucleic acid editing, nucleic acid splicing; It can be used in transport, tracking of target nucleic acids, isolation of target nucleic acids, visualization of target nucleic acids, and the like.

Aspects of the invention are also herein in in vitro, in vivo or ex vivo genomic engineering in prokaryotic or eukaryotic cells, eg, for altering or manipulating the expression of one or more genes or one or more gene products. Also includes the methods and uses of the compositions and systems described in the document.

In one embodiment, the invention provides a method of cleaving the target DNA. The method may include modifying the target DNA with a nucleic acid targeting complex that binds to the target DNA and results in cleavage of the target DNA. In certain embodiments, the nucleic acid targeting complexes of the invention can produce cleavages (eg, single-strand or double-strand breaks) in RNA sequences when introduced into cells. For example, the method can be used to cleave intracellular disease RNA. For example, an exogenous RNA template in which an upstream sequence and a downstream sequence are adjacent to the sequence to be integrated may be introduced into a cell. These upstream and downstream sequences share sequence similarity with both sides of the integration site in RNA. If desired, the donor RNA may be mRNA. The exogenous RNA template comprises the sequence to be incorporated (eg, mutant RNA). The integrated sequence may be an endogenous or exogenous sequence for the cell. Examples of sequences to be incorporated include RNA encoding a protein or non-encoding RNA (eg, microRNA). Thus, the built-in sequences can be operably linked to one or more suitable control sequences. Alternatively, the sequence to be incorporated may provide accommodation. Upstream and downstream sequences in the exogenous RNA template are selected to facilitate recombination between the RNA sequence of interest and the donor RNA. The upstream sequence is an RNA sequence that shares sequence similarity with the RNA sequence upstream of the target integration site. Similarly, the downstream sequence is an RNA sequence that shares sequence similarity with the RNA sequence downstream of the target integration site. Upstream and downstream sequences in the exogenous RNA template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the target RNA sequence. Preferably, the upstream and downstream sequences in the exogenous RNA template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target RNA sequence. In some methods, the upstream and downstream sequences in the exogenous RNA template have about 99% or 100% sequence identity with the target RNA sequence. Upstream or downstream sequences are from about 20 bp to about 2500 bp, eg, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700. It may include 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, exemplary upstream or downstream sequences have from about 200 bp to about 2000 bp, from about 600 bp to about 1000 bp, or more specifically from about 700 bp to about 1000 bp. In some methods, the exogenous RNA template may further include markers. Such markers can facilitate screening for target integration. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous RNA template of the present invention can be constructed using recombinant techniques (see, eg, Sambrook et al., 2001 and Ausubel et al., 1996). In the method of modifying a target DNA by incorporating an exogenous DNA template, a cleavage (for example, double-stranded or single-stranded cleavage) is introduced into the DNA sequence by a nucleic acid targeting complex, and this is performed by homologous recombination with the exogenous DNA template. When the cleavage is repaired, the template is integrated into the DNA target. The presence of double-strand breaks facilitates template incorporation. In another embodiment, the invention provides a method of modifying RNA expression in eukaryotic cells. The method comprises increasing or decreasing the expression of a target polynucleotide using a nucleic acid targeting complex that binds to DNA encoding RNA (eg, mRNA or pre-mRNA). In some methods, inactivating the target DNA can result in altered expression in the cell. For example, when a DNA targeting complex binds to an intracellular target sequence, the target DNA is inactivated so that the sequence is not transcribed, no encoded protein is produced, or the sequence is like a wild-type sequence. Will not work. For example, the sequence encoding the protein or microRNA can be inactivated to prevent the production of protein or microRNA or premicroRNA transcripts. The target DNA of the DNA targeting complex can be any DNA endogenous or exogenous to eukaryotic cells. For example, the target DNA may be DNA present in the nucleus of a eukaryotic cell. The target DNA may be a sequence encoding a gene product (eg mRNA or premRNA) encoding a gene product (eg protein) or a non-coding sequence (eg ncRNA, lncRNA, tRNA, or rRNA). Examples of target DNAs include sequences associated with biochemical signaling pathways, such as DNA associated with biochemical signaling pathways. Examples of target DNA include disease-related DNA. "Disease-related" DNA refers to any DNA that produces a transcript at an abnormal level or in an abnormal form in cells derived from affected tissue as compared to non-disease control tissue or cells. It may be DNA transcribed from a gene that becomes expressed at abnormally high levels; it may be DNA transcribed from a gene that becomes expressed at abnormally low levels, where changes in expression Correlates with the onset and / or progression of the disease. Disease-related DNA is also transcribed from genes with one or more mutations or genetic variations that are directly involved in the etiology of the disease or have linkage disequilibrium with one or more genes involved. Also refers to DNA. The translation product may be known or unknown, and may be at normal or abnormal levels. The target DNA of the DNA targeting complex can be any DNA endogenous or exogenous to the eukaryotic cell. For example, the target DNA may be DNA present in the nucleus of a eukaryotic cell. The target DNA may be a sequence encoding a gene product (eg, mRNA, premRNA, protein) or a non-coding sequence (eg, ncRNA, lncRNA, tRNA, or rRNA).

In some embodiments, the method can include the step of binding a nucleic acid targeting complex to the target DNA to cause cleavage of the target DNA, thereby modifying the target DNA, wherein the nucleic acid targeting complex. The body comprises a nucleic acid targeting effector protein that forms a complex with a guide RNA that hybridizes to the target sequence in said target DNA. In one aspect, the invention provides a method of modifying the expression of DNA or RNA in eukaryotic cells. In some embodiments, the method comprises binding a nucleic acid targeting complex to DNA, which causes an increase or decrease in the expression of the DNA; where the nucleic acid targeting complex is a guide RNA. Contains nucleic acid targeting effector proteins that form a complex with. Similar considerations and conditions apply to methods of modifying the target DNA as described above. In practice, these sampling, culturing and reintroduction options apply to all aspects of the invention. In one aspect, the invention provides a method of modifying target DNA in eukaryotic cells, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal, and modifying one or more cells. Culturing can be done in Exobibo at any stage. The one or more cells may be further reintroduced into a non-human animal or plant. For cells to be reintroduced, it is particularly preferred that the cells are stem cells.

In fact, in any aspect of the invention, the nucleic acid targeting complex may comprise a nucleic acid targeting effector protein that forms a complex with a guide RNA that hybridizes to a target sequence.

The present invention relates to the engineering and optimization of systems, methods and compositions used to control gene expression involving DNA sequence targeting related to nucleic acid targeting systems and their components. The advantage of this method is that the CRISPR system minimizes or avoids off-target binding and the resulting side effects. This is achieved using a system configured to have a high degree of sequence specificity for the target DNA.

With respect to the nucleic acid targeting complex or system, preferably the tracr sequence has one or more hairpins and is at least 30 nucleotides in length, at least 40 nucleotides in length, or at least 50 nucleotides in length; the crRNA sequence is at least 10 to 30 nucleotides in length. Yes, the nucleic acid targeting effector protein is a type II Cas9 effector protein.

Editing and Modification In one aspect, the present invention provides a method of using one or more elements of the CRISPR system. The CRISPR complex of the present invention provides an effective means of modifying a target polynucleotide. The CRISPR complex of the present invention has a wide range of usefulness, including modifying (eg, deleting, inserting, translocating, inactivating, activating) target polynucleotides of a wide variety of cell types. Therefore, the CRISPR complex of the present invention has wide applicability in, for example, gene therapy, drug screening, disease diagnosis, and prognosis determination. An exemplary CRISPR complex comprises a CRISPR enzyme that forms a complex with a guide sequence that hybridizes to a target sequence within a target polynucleotide. In certain embodiments, the guide sequence is linked to a direct repeat sequence.

DNA Cleavage and Repair The method comprises modifying the target polynucleotide using a CRISPR complex that binds to the target polynucleotide and results in cleavage of the target polynucleotide. Typically, the CRISPR complex of the invention, when introduced into a cell, produces a cleavage (eg, single-strand or double-strand cleavage) in the genomic sequence. For example, this method can be used to cleave disease genes in cells. Cleavage produced by the CRISPR complex can be repaired by repair processes such as the error prone non-homologous end joining (NHEJ) pathway or high fidelity homologous recombination repair (HDR). In these repair processes, exogenous polynucleotide templates can be introduced into the genomic sequence. In some methods, HDR processes are used to modify the genome sequence. For example, an exogenous polynucleotide template containing an upstream sequence and a sequence to be integrated adjacent to the downstream sequence is introduced into the cell. Upstream and downstream sequences share sequence similarity with both sides of the integration site on the chromosome. If desired, the donor polynucleotide is a delivery vehicle such as DNA, such as a DNA plasmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), viral vector, linear DNA fragment, PCR fragment, naked nucleic acid, or liposome or poroxamar. It may be a nucleic acid complexed with. The exogenous polynucleotide template contains the sequence to be integrated (eg, a mutated gene). The integrated sequence may be an endogenous or exogenous sequence for the cell. Examples of sequences to be incorporated include polynucleotides encoding proteins or non-encoding RNAs (eg, microRNAs). Thus, the built-in sequences can be operably linked to one or more suitable control sequences. Alternatively, the sequence to be incorporated may provide accommodation. Upstream and downstream sequences in the exogenous polynucleotide template are selected to facilitate recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with the genomic sequence upstream of the target integration site. Similarly, a downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the target integration site. Upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the target genomic sequence. Preferably, the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target genomic sequence. In some methods, the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the target genomic sequence. Upstream or downstream sequences are from about 20 bp to about 2500 bp, eg, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700. It may include 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, exemplary upstream or downstream sequences have from about 200 bp to about 2000 bp, from about 600 bp to about 1000 bp, or more specifically from about 700 bp to about 1000 bp. In some methods, the exogenous polynucleotide template may further comprise a marker. Such markers can facilitate screening for target integration. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous polynucleotide template of the present invention can be constructed using recombinant techniques (see, eg, Sambrook et al., 2001 and Ausube et al., 1996). In a method of modifying a target polynucleotide by incorporating an exogenous polynucleotide template, the CRISPR complex introduces a double-strand break into the genomic sequence, and homologous recombination with the exogenous polynucleotide template repairs this break. , The template is integrated into the genome. The presence of double-strand breaks facilitates template incorporation. In another embodiment, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. The method comprises increasing or decreasing the expression of the target polynucleotide using a CRISPR complex that binds to the polynucleotide. In some methods, inactivating the target polynucleotide can result in altered expression in the cell. For example, when the CRISPR complex binds to an intracellular target sequence, the target polynucleotide is inactivated so that the sequence is not transcribed, no encoded protein is produced, or the sequence is like a wild-type sequence. Will not work. For example, the sequence encoding the protein or microRNA can be inactivated to prevent the production of protein or microRNA or premicroRNA transcripts. In some methods, the control sequence can be inactivated so that it no longer functions as a control sequence. As used herein, "control sequence" refers to any nucleic acid sequence that provides transcription, translation, or contactability of the nucleic acid sequence. Examples of control sequences include promoters, transcription terminators, and enhancers are control sequences. The target polynucleotide of the CRISPR complex may be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide may be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or junk DNA). Examples of target polynucleotides include sequences associated with biochemical signaling pathways, such as genes or polynucleotides associated with biochemical signaling pathways. Examples of target polynucleotides include disease-related genes or polynucleotides. A "disease-related" gene or polynucleotide is any gene that produces a transcription or translation product at an abnormal level or in an abnormal form in cells derived from the affected tissue as compared to a non-disease control tissue or cell. Or refers to a polynucleotide. It may be a gene that becomes expressed at abnormally high levels; it may be a gene that becomes expressed at abnormally low levels, where changes in expression correlate with the development and / or progression of the disease. To do. Disease-related genes also refer to genes with one or more mutations or genetic variations that are directly involved in the cause of the disease or have linkage disequilibrium with one or more genes involved therein. The transcribed or translated product may be known or unknown, and may be at normal or abnormal levels. The target polynucleotide of the CRISPR complex may be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide may be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or junk DNA).

Gene editing or alteration of the target locus by Cas9; the single-strand break point at the HDR and template double-strand or one of the strands must be advantageously close enough to the target location for modification to occur. It doesn't become. In certain embodiments, the distance is 50, 100, 200, 300, 350 or 400 nucleotides or less. Although not desired to be constrained by theory, it is believed that the cleavage point must be close enough to the target location and that there be a cleavage point between the terminal recessions within the region subject to exonuclease-mediated removal. Since the template nucleic acid sequence can only be used to modify the sequence within the terminal section region, if the distance between the target position and the cleavage point is too large, the terminal section may not contain mutations. As a result, it may not be fixed.

In embodiments where the guide RNA and type II molecule, specifically Cas9 or its ortholog or homolog, preferably Cas9 nuclease, induces double-strand breaks to induce HDR-mediated modification, the cleavage site is 0-200 bp (eg, 0-200 bp). , 0-175, 0-150, 0-125, 0-100, 0-75, 0-50, 0-25, 25-200, 25-175, 25-150, 25-125, 25-100, 25 ~ 75, 25-50, 50-200, 50-175, 50-150, 50-125, 50-100, 50-75, 75-200, 75-175, 75-150, 75-125, 75-100bp ) Only away from the target position. In certain embodiments, the cleavage site is 0 to 100 bp (eg, 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp. ) Only away from the target position. In a further embodiment, to induce HDR-mediated modification, multiplex cleavage can be induced using two or more guide RNAs complexed with Cas9 or its ortholog or homolog.

The homology arm must extend at least to the extent of potential terminal recession so that the recessed single-strand overhang can find complementary regions within the donor template, for example. It doesn't become. Overall length can be limited by parameters such as plasmid size or virus packaging restrictions. In certain embodiments, the homology arm does not have to extend into the repeating element. Exemplary homology arm lengths include at least 50, 100, 250, 500, 750 or 1000 nucleotides.

The target location, as used herein, is type II, specifically Cas9 or its ortholog or homolog, preferably a site on the target nucleic acid or target gene (eg, chromosome) that is modified by a Cas9 molecule-dependent process. Point to. For example, the target position can be a modification of the target nucleic acid, Cas9 molecule cleavage, and modification of the target position by inducing a template nucleic acid, for example, modification. In certain embodiments, the target location can be a site between two nucleotides on the target nucleic acid to which one or more nucleotides are added, eg, between adjacent nucleotides. The target position can include one or more nucleotides that vary depending on the template nucleic acid, eg, are modified. In certain embodiments, the target location is within the target sequence (eg, the sequence to which the guide RNA binds). In certain embodiments, the target location is upstream or downstream of the target sequence (eg, the sequence to which the guide RNA binds).

Template nucleic acids, as the term is used herein, are combined with type II molecules, specifically Cas9 or its orthologs or homologs, preferably Cas9 molecules and guide RNA molecules, to alter the structure of the target location. Refers to a nucleic acid sequence that can be used. In certain embodiments, the target nucleic acid is typically modified to have some or all of the sequence of template nucleic acids in or near one or more cleavage sites. In certain embodiments, the template nucleic acid is single strand. In an alternative embodiment, the template nucleic acid is double-stranded. In certain embodiments, the template nucleic acid is DNA, eg, double-stranded DNA. In an alternative embodiment, the template nucleic acid is single-stranded DNA.

In certain embodiments, the template nucleic acid alters the structure of the target position by participating in homologous recombination. In certain embodiments, the template nucleic acid alters the sequence of target positions. In certain embodiments, the template nucleic acid results in the uptake of modified or non-naturally occurring bases into the target nucleic acid.

The template sequence can undergo cleavage-mediated or catalytic recombination with the target sequence. In certain embodiments, the template nucleic acid may comprise a sequence corresponding to a site on the target sequence that is cleaved by a Cas9-mediated cleavage event. In certain embodiments, the template nucleic acid is a first site on a target sequence that is cleaved in a first Cas9-mediated event and a second site on a target sequence that is cleaved in a second Cas9-mediated event. It may contain sequences corresponding to both.

In certain embodiments, the template nucleic acid is a sequence that alters the coding sequence of the sequence to be translated, eg, substitution of one amino acid in a protein product with another, eg, a mutation allele to a wild allele. Includes those that result in gene transformation, transformation of wild alleles into mutant alleles, and / or introduction of stop codons, insertion of amino acid residues, deletion of amino acid residues, or nonsense mutations. Can be done. In certain embodiments, the template nucleic acid can include sequences that result in changes in non-coding sequences, such as exons or 5'or 3'untranslated or untranscribed regions. Such changes include changes in control elements such as promoters, enhancers, and changes in cis or trans-acting control elements.

The structure of the target sequence may be changed by using a template nucleic acid having homology with the target position of the target gene. Template sequences can be used to alter undesired structures, such as undesired or mutated nucleotides. When the template nucleic acid is integrated, the activity of the positive control element decreases; the activity of the positive control element increases; the activity of the negative control element decreases; the activity of the negative control element increases; the expression of the gene decreases; the expression of the gene Increased; increased resistance to disorders or diseases; increased resistance to viral invasion; correction of mutations or changes in unwanted amino acid residues, conferral, increase, elimination or decrease of biological properties of gene products, such as enzymes Can include sequences that result in increased enzymatic activity of the virus, or increased ability of the gene product to interact with another molecule.

The template nucleic acid may contain sequences that result in sequence alterations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 nucleotides or more of the target sequence. In certain embodiments, the template nucleic acids are 20 ± 10, 30 ± 10, 40 ± 10, 50 ± 10, 60 ± 10, 70 ± 10, 80 ± 10, 90 ± 10, 100 ± 10, 110 ± 10, 120. It may be ± 10, 130 ± 10, 140 ± 10, 150 ± 10, 160 ± 10, 170 ± 10, 180 ± 10, 190 ± 10, 200 ± 10, 210 ± 10, or 220 ± 10 nucleotides in length. .. In certain embodiments, the template nucleic acids are 30 ± 20, 40 ± 20, 50 ± 20, 60 ± 20, 70 ± 20, 80 ± 20, 90 ± 20, 100 ± 20, 110 ± 20, 120 ± 20, 130. It may be ± 20, 140 ± 20, 150 ± 20, 160 ± 20, 170 ± 20, 180 ± 20, 190 ± 20, 200 ± 20, 210 ± 20, or 220 ± 20 nucleotides in length. In certain embodiments, the template nucleic acids are 10-1,000, 20-900, 30-800, 40-700, 50-600, 50-500, 50-400, 50-300, 50-200, or 50-. It is 100 nucleotides in length.

The template nucleic acid contains the following components: [5'homologous arm]-[substitution sequence]-[3'homologous arm]. The homology arm results in recombination to the chromosome and thus substitution by an undesired element such as a mutation or signature substitution sequence. In certain embodiments, the homologous arm is adjacent to the most distal amputation site. In certain embodiments, the 3'end of the 5'homologous arm is next to the 5'end of the substitution sequence. In certain embodiments, the 5'homologous arm is at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 5'from the 5'end of the substitution sequence. 900, 1000, 1500, or 2000 nucleotides can be extended. In certain embodiments, the 5'end of the 3'homologous arm is located next to the 3'end of the substitution sequence. In certain embodiments, the 3'homologous arm is at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 3'from the 3'end of the substitution sequence. 900, 1000, 1500, or 2000 nucleotides can be extended.

In certain embodiments, one or both homology arms can be shortened to avoid the inclusion of certain sequence repeat elements. For example, the 5'homology arm may be shortened to avoid array repeat elements. In other embodiments, the 3'homology arm may be shortened to avoid sequence repeat elements. In some embodiments, both the 5'and 3'homology arms may be shortened to avoid the inclusion of certain sequence repeat elements.

In certain embodiments, template nucleic acids for correcting mutations can be designed to be used as single-strand oligonucleotides. When using single-strand oligonucleotides, the 5'and 3'homologous arms extend to about 200 base pair (bp) length, eg, at least 25, 50, 75, 100, 125, 150, 175, or 200 bp length. It can be a range.

DNA repair and NHEJ
In certain embodiments, nuclease-induced non-homologous end joining (NHEJ) can be used to target gene-specific knockouts. The nuclease-induced NHEJ can also be used to remove (eg, delete) a sequence within a gene of interest. In general, NHEJ repairs double-strand breaks in DNA by joining their ends together; however, in general, the original sequences are restored when they were initially formed by double-strand breaks. Only if the two compatible ends are completely ligated exactly as they are. Since the DNA ends of double-strand breaks often undergo enzymatic processing, nucleotide additions or removals to one or both strands occur before those ends are reconnected. This results in the presence of insertion and / or deletion (indel) mutations in the DNA sequence of the NHEJ repair site. Two-thirds of these mutations typically alter the reading frame, thus producing non-functional proteins. In addition, mutations that insert or delete many sequences while maintaining a reading frame can disrupt protein functionality. This is locus-dependent because mutations in critical functional domains are expected to be less tolerant than mutations in non-critical regions of the protein. The indel mutations produced by NHEJ are inherently unpredictable; however, due to the small microhomology region, specific indel sequences are preferred at a given cleavage site, resulting in large proportions within the population. Occupy. Deletion lengths can vary widely; most commonly in the range of 1-50 bp, but can be well over 50 bp, for example well over about 100-200 bp. .. Insertions tend to be shorter and often contain short overlaps of sequences that directly surround the cut site. However, it is possible to obtain large insertions, in which case the inserted sequences often reach into plasmid DNA present in other regions of the genome or cells.

Since NHEJ is a mutagenic process, it can also be used for deletion of small sequence motifs, unless specific final sequence generation is required. When double-strand breaks target the vicinity of short target sequences, deletion mutations caused by NHEJ repair often involve unwanted nucleotides, thus eliminating them. For deletion of larger DNA segments, the introduction of two double-strand breaks, one on each side of the sequence, can result in NHEJ between their ends and removal of the entire intervening sequence. Both of these techniques can be used for deletion of specific DNA sequences; however, the error-prone nature of NHEJ can still produce indel mutations at the repair site.

Double-stranded cleave type II molecule, specifically Cas9 or its ortholog or homolog, preferably Cas9 molecule and single-stranded, or nickase type II molecule, specifically Cas9 or its ortholog or homolog, preferably Cas9 molecule Both can be used to generate NHEJ-mediated indels in the methods and compositions described herein. NHEJ-mediated indels that target a gene, eg, the coding region of the gene of interest, eg, the initial coding region, can be used to knock out (ie, abolish) the gene of interest. For example, the initial coding region of the gene of interest is within 500 bp of the sequence immediately following the transcription initiation site, the sequence within the first exon of the coding sequence, or the transcription initiation site (eg, 500, 450, 400, 350, 300, 250, 200, 150, 100 or less than 50 bp) sequences.

In certain embodiments, the guide RNA and type II molecule, specifically Cas9 or its ortholog or homolog, preferably Cas9 nuclease, produces a double-strand break to induce an NHEJ-mediated indel, the guide RNA is at the target location It can be configured to locate one double-strand break in close proximity to the nucleotide. In certain embodiments, the cleavage site is 0 to 500 bp from the target position (eg, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7 from the target position. , 6, 5, 4, 3, 2 or less than 1 bp).

One embodiment in which two guide RNAs that form a complex with a type II molecule, specifically Cas9 or its ortholog or homolog, preferably Cas9 nickase, induce two single-strand breaks to induce NHEJ-mediated indels. In, the two guide RNAs can be configured to position two single-strand breaks to result in NHEJ repair of the nucleotide at the target location.

Delivery of Functional Effectors Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminates expression by mutating the gene at the DNA level, CRISPR-Cas knockdown uses artificial transcription factors to temporarily eliminate gene expression. It is possible to reduce it. Mutation of key residues in both DNA cleavage domains of the Cas9 protein produces catalytically inactive Cas9. The catalytically inactive Cas9 forms a complex with the guide RNA and localizes to the DNA sequence identified by the targeting domain of the guide RNA, however, it does not cleave the target DNA. Fusing an inactive Cas9 protein with an effector domain, such as a transcriptional repressor domain, allows the effector to be recruited to any DNA site identified by the guide RNA. In certain embodiments, Cas9 may be fused to a transcriptional repression domain and recruited to the promoter region of the gene. Especially with respect to gene suppression, it is contemplated herein that blocking the binding site of an endogenous transcription factor can help downregulate gene expression. In another embodiment, the inert Cas9 may be fused with a chromatin-modified protein. Changes in chromatin status can result in decreased expression of the target gene.

In certain embodiments, the guide RNA molecule is unknown or known to be able to control the expression of known transcriptional response elements (eg, promoters, enhancers, etc.), known upstream activation sequences, and / or target DNA. Sequences of function can be targeted.

In some methods, inactivating the target polynucleotide can result in altered expression in the cell. For example, when the CRISPR complex binds to an intracellular target sequence, the target polynucleotide is inactivated so that the sequence is not transcribed, no encoded protein is produced, or the sequence is like a wild-type sequence. Will not work. For example, a protein or microRNA coding sequence can be inactivated to prevent protein production.

In certain embodiments, the CRISPR enzyme comprises one or more mutations selected from the group consisting of D917A, E1006A and D1225A, and / or one or more mutations are in the RuvC domain of the CRISPR enzyme. Or other mutations as discussed herein. In some embodiments, the CRISPR enzyme has one or more mutations in the catalytic domain, where when transcribed, the direct repeat sequence forms a single stem-loop and the guide sequence to the target sequence. Derives sequence-specific binding of the CRISPR complex, where the enzyme further comprises a functional domain. In some embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is a SID, or a concatemer of the SID (eg, SID4X). In some embodiments, the functional domain is an epigenetic modified domain, providing an epigenetic modified enzyme. In some embodiments, the functional domain is the activation domain, which may be the P65 activation domain.

Functional effector (domain)
Gene editing may be performed with Cas9 of the present invention. Cas9 may be inactivated and fused to one or more functional domains (effectors).

In some embodiments, the functional domains are transposase domain, integrase domain, recombinase domain, resolverase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain. , Histone deacetylase domain, nuclease domain, repressor domain, activator domain, transcriptional regulatory protein (or transcription complex recruitment) domain, cell uptake activity related domain, nucleic acid binding domain, antibody presentation domain, histone modifier, histone modification Enzyme recruiters; histone modifying enzyme inhibitors, histone methyltransferases, histone demethylases, histone kinases, histone phosphatases, histone ribosylases, histone deribosyrase, histone ubiquitinase, histone deubiquitinase, histone biotinase and It can be selected from the group consisting of histone tail proteases.

In some preferred embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is a SID, or a concatemer of the SID (eg, SID4X). In some embodiments, the functional domain is an epigenetic modified domain, providing an epigenetic modified enzyme. In some embodiments, the functional domain is the activation domain, which may be the P65 activation domain.

Gene targeting in non-dividing cells (neurons and muscles) For example, homologous recombination (HR) is generally suppressed during the G1 phase of the cell cycle, so non-dividing (especially non-dividing, fully differentiated) cell types are muscle cells and It is a problem in gene targeting or genome engineering, especially including neurons. However, while studying the mechanisms by which cells control the normal DNA repair system, Orthwein et al. Have reported on a previously unknown switch that keeps HR "off" in non-dividing cells, and they have devised a strategy to switch this switch back on. Orthwein et al. (Daniel Durocher's lab at the Mount Sinai Hospital in Ottawa, Canada, reporting in Nature 16142, published online 9 Dec 2015) can release the suppression of HR, kidney (293T) and kidney (293T) It shows that gene targeting was successful in both cells. Tumor suppressors BRCA1, PALB2 and BRAC2 are known to promote DNA DSB repair by HR. They found that the formation of a complex of BRCA1 and PALB2-BRAC2 was dependent on the ubiquitin site on PALB2 (eg, the effect of E3 ubiquitin ligase on that site). This E3 ubiquitin ligase is composed of KEAP1 (PALB2 interacting protein) complexed with Karin-3 (CUL3) -RBX1. Ubiquitination of PALB2 suppresses its interaction with BRCA1 and is countered by the deubiquitinating enzyme USP11, which itself is under cell cycle control. To induce homologous recombination in G1, DNA-terminated recession, as measured by a number of methods, including the CRISPR-Cas9-based gene targeting assay (expressed from the pX459 vector) directed at USP11 or KEAP1. It suffices if the BRCA1-PALB2 interaction is restored with activation. However, when either KEAP1 depletion or expression of the PALB2-KR mutant was used to restore BRCA1-PALB2 interactions in recession-competent G1 cells, a robust increase in gene targeting events was observed.

Therefore, reactivation of HR in non-dividing, fully differentiated cell types, including cells, especially muscle cells and especially neurons, is preferred in some embodiments. In some embodiments, facilitating the BRCA1-PALB2 interaction is preferred in some embodiments. In some embodiments, the target cell is a non-dividing cell. In some embodiments, the target cell is a neuron or muscle cell. In some embodiments, the target cells are targeted in vivo. In some embodiments, the cells are in G1 phase and HR is suppressed.

In some embodiments, it is preferred to use KEAP1 depletion, eg inhibition of expression of KEAP1 activity. KEAP1 depletion is described, for example, in Orthwein et al. As shown in, it can be realized by siRNA. Alternatively, expression of the PALB2-KR mutant (which lacks all eight Lys residues of the BRCA1 interaction domain) is preferred, either in combination with KEAP1 depletion or alone.

PALB2-KR interacts with BRCA1 regardless of cell cycle position. Thus, especially in G1 cells, facilitation or recovery of BRCA1-PALB2 interactions, in some embodiments, is particularly problematic for target cells to be non-dividing or for removal and recovery (exovibo gene targeting). For example, it is preferable when it is a neuron or a muscle cell. KEAP1 siRNA is preferred and is available from ThermoFisher.

In some embodiments, the BRCA1-PALB2 complex is delivered to G1 cells as either a protein complex, a fusion protein, a polynucleotide encoding BRCA1 and PALB2, or a polynucleotide encoding a BRCA1-PALB2 fusion protein. You may. Such polynucleotides may be delivered, for example, under the control of a suitable promoter, simultaneously and optionally in the same vector or vector system as the CRISPR protein, or separately, as described herein. It may be delivered. Another possibility of promoting HR in non-dividing, fully differentiated cell types, including myocytes and especially neurons, is direct delivery of PALB2 (using Cas9 fused to a molecule that is compatible with PALB2); / Or direct delivery of BRCA2 (using Cas9 fused to an affinity molecule for BRCA2) can be mentioned.

In some embodiments, deubiquitination of PALB2 may be promoted, for example by increasing the expression or activity of the deubiquitinating enzyme USP11. Therefore, it is envisioned that, in some embodiments, it may be possible to provide a construct that promotes or upregulates the expression or activity of the deubiquitinating enzyme USP11.

Knockdown of CRL4 can also be used to inactivate KEAP1 or reduce its activity. For example, CRL4 siRNA may be used. Alternatively, MLN4924 (a pan-CRL inhibitor) can also be used to inactivate KEAP1.

A knockout of 53BP is also particularly preferred, as it has been suggested that this is necessary for HDR activation (shown in Orthwein et al). Knockout of 53BP can also be achieved by siRNA.

Activation of DNA recession (creating a 3'overhang on each side of the DNA double-strand break) was also essential for activation of HR in G1. This was done by delivering the ORF of the gene CtIP (or SAE2) with a mutation (T847E) that mimics activated phosphorylation. Applicants now hypothesize that this requirement can be circumvented by introducing a 3'overhang using Cas9 double nickase (Hsu et al). Therefore, introducing a 3'overhang using Cas9 double nickase in this way is preferred for targeting non-dividing, fully differentiated cell types, including myocytes and especially neurons.

In one embodiment, the invention provides a method of cleaving a target polynucleotide. The method comprises using a CRISPR complex that binds to the target polynucleotide to cause cleavage of the target polynucleotide. Typically, the CRISPR complexes of the invention produce cleavages (eg, single-strand or double-strand breaks) in the genomic sequence when introduced into cells. For example, the method can be used to cleave intracellular disease genes.

Cleavage produced by the CRISPR complex can be repaired by repair processes such as the Errorpron Nonhomologous End Binding (NHEJ) pathway or High Fidelity Homologous Recombination Repair (HDR). In these repair processes, exogenous polynucleotide templates can be introduced into the genomic sequence. In some methods, HDR processes are used to modify the genome sequence. For example, an exogenous polynucleotide template containing an upstream sequence and a sequence to be integrated adjacent to the downstream sequence is introduced into the cell. Upstream and downstream sequences share sequence similarity with both sides of the integration site on the chromosome.

If desired, the donor polynucleotide is a delivery vehicle such as DNA, such as a DNA plasmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), viral vector, linear DNA fragment, PCR fragment, naked nucleic acid, or liposome or poroxamar. It may be a nucleic acid complexed with.

The exogenous polynucleotide template contains the sequence to be integrated (eg, a mutated gene). The integrated sequence may be an endogenous or exogenous sequence for the cell. Examples of integration sequences to be incorporated include polynucleotides encoding proteins or non-encoding RNAs (eg, microRNAs). Thus, the built-in sequence may be operably linked to one or more suitable control sequences. Alternatively, the built-in sequence to be incorporated may provide accommodation.

Upstream and downstream sequences in the exogenous polynucleotide template are selected to facilitate recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with the genomic sequence upstream of the target integration site. Similarly, a downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the target integration site. Upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the target genomic sequence. Preferably, the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target genomic sequence. In some methods, the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the target genomic sequence.

Upstream or downstream sequences are from about 20 bp to about 2500 bp, eg, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700. It may include 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, exemplary upstream or downstream sequences have from about 200 bp to about 2000 bp, from about 600 bp to about 1000 bp, or more specifically from about 700 bp to about 1000 bp.

In some methods, the exogenous polynucleotide template may further comprise a marker. Such markers can facilitate screening for target integration. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous polynucleotide template of the present invention can be constructed using recombinant techniques (see, eg, Sambrook et al., 2001 and Ausube et al., 1996).

In an exemplary method of modifying a target polynucleotide by incorporating an exogenous polynucleotide template, the CRISPR complex introduces a double-strand break into the genomic sequence and homologous recombination with the exogenous polynucleotide template repairs this break. When done, the template is integrated into the genome. The presence of double-strand breaks facilitates template incorporation.

In another embodiment, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. The method comprises increasing or decreasing the expression of the target polynucleotide using a CRISPR complex that binds to the polynucleotide.

In some methods, inactivating the target polynucleotide can result in altered expression in the cell. For example, when the CRISPR complex binds to an intracellular target sequence, the target polynucleotide is inactivated so that the sequence is not transcribed, no encoded protein is produced, or the sequence is like a wild-type sequence. Will not work. For example, a sequence encoding a protein or microRNA can be inactivated to prevent protein production.

In some methods, the control sequence can be inactivated so that it no longer functions as a control sequence. As used herein, "control sequence" refers to any nucleic acid sequence that provides transcription, translation, or contactability of the nucleic acid sequence. Examples of control sequences include promoters, transcription terminators, and enhancers are control sequences. The inactivated target sequence can be a deletion mutation (ie, deletion of one or more nucleotides), an insertion mutation (ie, insertion of one or more nucleotides), or a nonsense mutation (ie, termination codon). Can include replacement of a single nucleotide with another nucleotide such as which is introduced). In some methods, inactivating the target sequence results in a "knockout" of the target sequence.

Illustrative Use of CRISPR Cas Systems The present invention encodes non-naturally occurring or engineered compositions, or components of said compositions, used to modify target cells in vivo, ex vivo or in vitro. Provided is a vector or delivery system containing one or more polynucleotides, or one or more polynucleotides encoding the constituents of the composition, and after modification, the progeny or cell line of the cell modified by CRISPR is altered. It can be done in a way that the cells are altered to retain the phenotype. The modified cells and progeny can be part of a multicellular organism such as a plant or animal in which the CRISPR system has been applied ex vivo or in vivo to the desired cell type. The CRISPR invention can be a therapeutic method. Therapeutic treatment methods may include gene or genome editing, or gene therapy.

Modification of Target by CRISPR-Cas System or Complex In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal, and modifying one or more cells. Culturing can be done in Exobibo at any stage. The one or more cells may be further reintroduced into a non-human animal or plant. For cells to be reintroduced, it is particularly preferred that the cells are stem cells.

In some embodiments, the method comprises attaching the CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex is: Contains a CRISPR enzyme that hybridizes to or hybridizes to a target sequence within the target polynucleotide, the guide sequence is linked to a tracr mate sequence, and then the tracr mate sequence It hybridizes to the tracr sequence.

In one aspect, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises the step of binding a CRISPR complex to a polynucleotide that causes an increase or decrease in expression of the polynucleotide; where the CRISPR complex is said to be the poly. It contains a CRISPR enzyme that hybridizes to or is hybridized to a target sequence within a nucleotide and is complexed with a guide sequence, the guide sequence being linked to a tracr mate sequence, and then the tracr mate sequence hybridizing to the tracr sequence. To soy. Similar considerations and conditions apply to methods of modifying the target polynucleotide as described above. In practice, these sampling, culturing and reintroduction options apply to all aspects of the invention.

In fact, in any aspect of the invention, the CRISPR complex can include a CRISPR enzyme that hybridizes to or forms a complex with a guide sequence capable of hybridizing to the target sequence, wherein the guide sequence is a tracr mate sequence. The tracr mate sequence may then hybridize to the tracr sequence. Similar considerations and conditions apply to methods of modifying the target polynucleotide as described above.

Thus, it comprises at least one modification of any of the non-naturally occurring CRISPR enzymes described herein, whereby the enzyme has certain enhanced abilities. In particular, any of these enzymes is capable of forming a CRISPR complex with a guide RNA. During the formation of such a complex, the guide RNA is capable of binding to the target polynucleotide sequence and the enzyme is capable of modifying the target locus. In addition, the enzymes in the CRISPR complex have a reduced ability to modify one or more off-target loci when compared to unmodified enzymes.

In addition, the modified CRISPR enzymes (emzyme) described herein include enzymes that have an increased ability to modify one or more target loci when compared to non-modified enzymes in the CRISPR complex. Such a function may be provided separately from, or in combination with, the above-mentioned function of reducing the ability to modify one or more off-target loci. The CRISPR enzyme as described herein, such as any such enzyme in combination with any activity provided by one or more associated heterologous functional domains, any further mutation that reduces nuclease activity, etc. May be provided with any of the further modifications to.

In an advantageous embodiment of the invention, the ability to modify one or more off-target loci when compared to an unmodified enzyme and the ability to modify one or more target loci when compared to an unmodified enzyme. A modified CRISPR enzyme (emzyme) with an increase is provided. In combination with further modifications to the enzyme, a significant increase in specificity can be achieved. For example, a combination of such an advantageous embodiment and one or more additional mutations is provided, where the one or more additional mutations are in one or more catalytically active domains. Such additional catalytic mutations may confer nickase function as described in detail elsewhere herein. In such an enzyme, the specificity is improved in terms of enzyme activity, so that the specificity can be enhanced.

Modifications that reduce the off-target effect and / or enhance the on-target effect as described above are performed on amino acid residues located in the positively charged region / groove between the RuvC-III domain and the HNH domain. Can be It is understood that any of the functional effects described above can be achieved by modifying the amino acids in the groove as described above, but can also be achieved by modifying the amino acids adjacent to or outside the groove. Will be done.

Further features that can be engineered to result in a modified CRISPR enzyme as described herein include: 1. 1. A modified CRISPR enzyme that disrupts DNA: protein interactions without affecting protein tertiary or secondary structure. This includes residues that make contact with any part of the RNA: DNA double strand. 2. 2. A modified CRISPR enzyme that weakens the protein-protein interactions that Cas9 carries to take the conformation essential for nuclease cleavage in response to DNA binding (on or off-target). For example: A modification that slightly inhibits the nuclease conformation of the HNH domain (located in fragile phosphate), but still allows it. 3. 3. A modified CRISPR enzyme that enhances protein-protein interactions that hold Cas9 in a conformation that inhibits nuclease activity in response to DNA binding (on or off-target). For example: A modification that stabilizes the HNH domain in a conformation that keeps it away from the fragile phosphate. Any such additional enhancement may be provided in combination with any other modification to the CRISPR enzyme as described in detail elsewhere herein.

Any of the enhanced functions described herein can be added to any CRISPR enzyme, such as the Cas9 enzyme. The Cas9 enzymes described herein are derived from the Cas9 enzymes of Streptococcus pyogenes (S. pyogenes) and Staphylococcus aureus (S. aureus). However, it will be appreciated that any of the functions described herein can be engineered to Cas9 enzymes from other orthologs, including chimeric enzymes containing fragments from multiple orthologs. For examples of such orthologs, see, for example, FIGS. 8 and 9 as described herein.

The present invention uses nucleic acids to bind a target DNA sequence. This is advantageous because nucleic acids are much easier and cheaper to make than proteins, and their specificity can be varied depending on the length of stretch for which homology is required. For example, it is not necessary to arrange a plurality of fingers in a complicated three-dimensional manner. The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used synonymously. These terms refer to nucleotides in polymer form of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci defined by linkage analysis (one locus), exons, introns, messenger RNA ( mRNA), transfer RNA, ribosome RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, Isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. The term also includes nucleic acid-like structures having a synthetic backbone, eg, Eckstein, 1991; Baserga et al. , 1992; Milligan, 1993; International Publication No. 97/03211 Pamphlet; International Publication No. 96/39154 Pamphlet; Mata, 1997; Stratus-Sokuup, 1997; and Samstag, 1996. Polynucleotides can include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be imparted before or after assembling the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. As used herein, the term "wild type" is a term in the art understood by those skilled in the art, as naturally occurring as distinct from mutants or mutant forms. Means a typical form of organism, strain, gene, or characteristic. The "wild type" can be the baseline. As used herein, the term "mutant" shall be construed to mean the presentation of a quality with a pattern that deviates from what is naturally occurring. The terms "non-naturally occurring" or "engineered" are used synonymously to indicate human intervention. When the term refers to a nucleic acid molecule or polypeptide, the nucleic acid molecule or polypeptide is at least substantially substantially one other component that is naturally associated with it in nature and is as found in nature. "Complementarity", which means not included in, means that a nucleic acid forms one or more hydrogen bonds with another nucleic acid sequence by either conventional Watson-Crick base pairing or other non-conventional pairing. Refers to the ability to do. "Complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick base pairing or other non-conventional types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5, 6, 7 out of 10). , 8, 9, and 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). By "fully complementary" is meant that all contiguous residues of a nucleic acid sequence are hydrogen bonded to the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 over 24, 25, 30, 35, 40, 45, 50 nucleotides, or more regions. Refers to the degree of complementarity that is%, 99%, or 100%, or refers to two nucleic acids that hybridize under stringent conditions. As used herein, the "stringent condition" of hybridization is that a nucleic acid that is complementary to a target sequence preferentially hybridizes to the target sequence and substantially hybridizes to a non-target sequence. Refers to conditions that do not hybridize. Stringent conditions are generally sequence-dependent and depend on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions, Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay" , Elsevier, N. et al. Y. It is detailed in. When referring to polynucleotide sequences, complementary or partially complementary sequences are also envisioned. These are preferably those that are hybridizable with the reference sequence under highly stringent conditions. In general, for maximizing hybridization rates, relatively low stringency hybridization conditions (about 20-25 ° C. below the thermal melting point ( _Tm )) are selected. T _m is the temperature at which 50% of a particular target sequence hybridizes to a fully complementary probe in a solution of specified ionic strength and pH. Generally, if at least about 85% nucleotide complementarity of the hybridized sequence is required, highly stringent wash conditions are selected to be about 5-15 ° C. below _Tm . If at least about 70% nucleotide complementarity of the hybridized sequence is required, moderately stringent wash conditions are selected to be about 15-30 ° C below _Tm . Highly acceptable (very low stringency) wash conditions can be as much as 50 ° C. below _Tm, allowing high levels of mismatch between hybridized sequences. Those skilled in the art will also influence the results of detectable hybridization signals from specific levels of homology between the target and probe sequences, as well as other physical and chemical parameters during the hybridization and washing steps. You will recognize that it can be changed. Preferred highly stringent conditions are incubation at 42 ° C. in 50% formamide, 5 × SSC, and 1% SDS, or incubation at 65 ° C. in 5 × SSC and 1% SDS, and 0.2 × SSC and Includes washing at 65 ° C. in 0.1% SDS. “Hybridization” refers to a reaction in which one or more polynucleotides react to form a stable complex by hydrogen bonding between bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex can include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction can constitute a step with a larger process, such as initiation of PCR or enzymatic cleavage of the polynucleotide. A sequence that is hybridizable with a given sequence is referred to as the "complement" of that given sequence. As used herein, the term "genomic locus" or "locus" (plural locus) is a particular location of a gene or DNA sequence on a chromosome. A "gene" refers to a sequence of DNA or RNA encoding a polypeptide, or an RNA strand that plays a functional role in an organism and is therefore a molecular genetic unit in the organism. For the purposes of the present invention, a gene can be considered to contain a region that regulates the production of a gene product (whether or not such regulatory sequences are flanked by coding and / or transcriptional sequences). Therefore, genes are not necessarily limited to translation regulatory sequences such as promoter sequences, terminators, ribosome binding sites and intrasequence ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites and loci. A control area is included. As used herein, "genome locus expression" or "gene expression" is the process by which information from a gene is used to synthesize a functional gene product. The product of gene expression is often a protein, but for non-protein coding genes such as the rRNA or tRNA genes, the product is a functional RNA. The process of gene expression is used by all known life-eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), and viruses to produce functional products for survival. As used herein, "expression" of a gene or nucleic acid includes not only cellular gene expression, but also transcription and translation of one or more nucleic acids in the cloning system and any other context. .. As used herein, "expression" is also the process by which a polynucleotide is transcribed from a DNA template (such as to mRNA or other RNA transcript) and / or the transcribed mRNA followed by a peptide, polypeptide. , Or the process of being translated into protein. Transcripts and encoded polypeptides are collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells. The terms "polypeptide", "peptide" and "protein" are used herein synonymously to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also include modified amino acid polymers such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including optical isomers of both glycine and D or L, and amino acid analogs and peptide mimetics. included. As used herein, the term "domain" or "protein domain" refers to a portion of a protein sequence that can exist and function independently of the remaining protein strands. As described in aspects of the invention, sequence identity is related to sequence homology. Homology comparisons may be made by eye or, more commonly, with the help of readily available sequence comparison programs. These commercially available computer programs can calculate the percent (%) homology between two or more sequences, as well as the sequence identity shared by two or more amino acid or nucleic acid sequences. .. In some preferred embodiments, the capping region of dTALE described herein has a sequence that is at least 95% identical or shares the same identity as the capping region amino acid sequence provided herein. .. Sequence homology can be determined by any of a number of computer programs known in the art, such as BLAST or FASTA. Computer programs suitable for performing such alignments are the GCG Wisconsin Bestfit Package (University of Wisconsin, USA; Deverex et al., 1984, Nucleic Acids Research 12).
: 387). Examples of other software capable of performing sequence comparisons include, but are not limited to, BLAST packages (see Ausubel et al., 1999 supra-Chapter 18), FASTA (Atschul et al., 1990, J. Mol). Biol., 403-410), and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online search (see Ausubel et al., 1999 supra, pp. 7-58-7-60). However, it is preferable to use the GCG Bestfit program. Percentage (%) sequence homology may be calculated for contiguous sequences, i.e., aligning one sequence with the other, each amino acid or nucleotide in one sequence with the corresponding amino acid or nucleotide in the other sequence. Each residue is directly compared. This is called a "no gap" alignment. Such gapless alignment is performed on a relatively small number of residues. This is a very simple and consistent method, but cannot take into account, for example, that subsequent amino acid residues are out of alignment due to one insertion or deletion in the same sequence pair. Therefore, global alignment can potentially significantly reduce% homology. As a result, most sequence comparison methods are designed to generate optimal alignments that take into account possible insertions or deletions without undue penalties for the overall homology or identity score. To. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology or identity. However, these more complex methods assign a "gap penalty" to each gap that appears in the alignment, so for the same number of the same amino acids, a sequence alignment with as few gaps as possible (between two comparable sequences). (Reflecting high relevance) can achieve higher scores than sequences with many gaps. Typically, "Affinity gap costs" are used that impose a relatively high cost on the existence of a gap and reduce the penalty for each subsequent residue in that gap. This is the most commonly used gap scoring system. High gap penalties can, of course, produce optimized alignments with smaller gaps. In many alignment programs, it is possible to change the gap penalty. However, when using software for sequence comparison, it is preferable to use the default value. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for the gap and -4 for each extension. Therefore, in order to calculate the maximum% homology, it is necessary to first consider the gap penalty to generate the optimum alignment. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (Deverex et al., 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, BLAST package (Ausubel et al., See ^{1999 Short Protocols in Molecular Biology, 4} th Ed.-Chapter 18), FASTA (Altschul Et al., 1990 J. Mol. Biol. 403-410), and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online search (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pp. 7-58-7-60). However, for some applications it is preferred to use the GCG Bestfit program. A novel tool called BLAST 2 Technologies is also available for comparison of protein and nucleotide sequences (FEMS Microbiol Lett. 1999 174 (2): 247-50; FEMS Microbiol Lett. 1999 177 (1): 187-8 and See the website of the National Center for Biotechnology Information on the website of the National Institutes for Health (see the website of the National Center for Biotechnology Information). The final% homology can be measured in terms of identity, but the alignment process itself is not typically based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLASTUM62 matrix-the default matrix of the BLAST program suite. The GCG Wisconsin program generally uses either the official default values or, if available, a custom symbol comparison table (see the user manual for further details). For some applications, it is preferable to use official default values for GCG packages, or for other software, use default matrices such as BLOSUM62. Alternatively, the percentage homology is calculated using the multiple alignment function of DNASIS ™ (Hitachi Software) based on the same algorithm as Clustal (Higgins DG & Sharp PM (1988), Gene 73 (1), 237-244). You may. Once the software has generated the optimal alignment, it is possible to calculate% homology, preferably% sequence identity. Software typically does this as part of a sequence comparison and produces numerical results. The sequence can also have deletions, insertions or substitutions of amino acid residues that result in silent changes resulting in functionally equivalent substances. Planned amino acid substitutions may be made based on similarities in amino acid properties (such as residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties), thus making amino acids functional. It is useful to put them together in various groups. Amino acids may be grouped based solely on the properties of their side chains. However, it is even more useful to include mutation data as well. The set of amino acids thus obtained appears to be conserved for structural reasons. These pairs can be described in the form of Venn diagrams (Livingstone CD and Barton GJ (1993) "Protein sequence alignment: a strategy for the hierarchical". analog. -218). Conservative substitutions can be made, for example, according to the table below, which describes the generally accepted Venn diagram amino acid classification.

In embodiments of the invention, both possible homologous substitutions (substitutions and revisions) here mean the mutual exchange of existing amino acid residues or nucleotides with alternative residues or nucleotides. In the case of amino acids, it contains sequences (both polynucleotides or polypeptides) that can contain substitutions of the same type, such as basics, acidics, polarities, etc. Non-homologous substitution, that is, substitution from one class to another class of residue, ornithine (hereinafter referred to as Z), ornithine diaminobutyrate (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O). ), Substitutions involving the uptake of unnatural amino acids such as pyriylalanine, thienylalanine, naphthylalanine and phenylglycine can also occur. A variant amino acid sequence is preferably inserted between any two amino acid residues of a sequence containing an amino acid spacer such as a glycine or β-alanine residue as well as an alkyl group such as a methyl group, an ethyl group or a propyl group. Spacer group may be included. Yet another form, which involves the presence of one or more amino acid residues in the peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, the "peptoid form" is used to refer to a variant amino acid residue in which the α-carbon substituent is not on the α-carbon, but rather on the nitrogen atom of the residue. Methods for preparing peptides in the peptoid form are known in the art (eg, Simon RJ et al., PNAS (1992) 89 (20), 9637-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134).

Homology modeling: Corresponding residues in other Cas9 orthologs are described in Zhang et al. , 2012 (Nature; 490 (7421): 556-60) and Chen et al. , 2015 (PLoS Comput Biol; 11 (5): e100248), can be identified by a computational protein-protein interaction (PPI) method that predicts interactions mediated by the domain-motif interface. PrePPI, a structure-based PPI prediction method, combines structural evidence with non-structural evidence using a Bayesian statistical framework. This method involves pairing query proteins and using structural alignments to identify structural representations that correspond to either of their experimentally determined structures or homology models. Structural alignment is also used to identify both near and far adjacent structures by considering global and local geometric relationships. Whenever two adjacent structures of a structural representation form a complex reported in a protein databank, this defines a template for modeling the interaction between these two query proteins. The model of the complex is created by superimposing representative structures on the corresponding adjacent structures in the mold. This method is described in Dey et al. , 2013 (Prot Sci; 22: 359-66).

For the purposes of the present invention, amplification means any method using primers and polymerases capable of replicating the target sequence with reasonable fidelity. Amplification can be performed by natural or recombinant DNA polymerases such as TaqGold ™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. The preferred amplification method is PCR.

In certain embodiments, the present invention relates to vectors. As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. This is a replicon, such as a plasmid, phage, or cosmid, on which another DNA segment can be inserted to result in replication of the inserted segment. In general, the vector is replicable when associated with the appropriate control element. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; free-ended (eg, circular) nucleic acid molecules containing one or more free ends; DNA, Nucleic acid molecules containing RNA, or both; and other types of polynucleotides known in the art. Certain vectors are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where the vector is of packaging into a virus (eg, retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus (AAV)). There is a virus-derived DNA or RNA sequence for this. Viral vectors also include polynucleotides carried by the virus for transfection into host cells. A particular vector is capable of self-renewal in the host cell into which it is introduced (eg, a bacterial vector with a bacterial origin of replication and an episomal mammalian vector). Other vectors (eg, non-episome mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. In addition, a particular vector can guide the expression of a gene to which it is operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

The recombinant expression vector can contain a form of nucleic acid of the invention suitable for expression of the nucleic acid in the host cell, that is, one in which the recombinant expression vector is operably linked to the nucleic acid sequence to be expressed. It is meant to include the above regulatory elements, which may be selected based on the host cell used for expression. Within the scope of the recombinant expression vector, "operably linked" allows expression of the nucleotide sequence (eg, in an in vitro transcription / translation system, or in the host cell when the vector is introduced into the host cell). It is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements. Regarding recombination and cloning methods, U.S. Patent Application No. 10 / 815,730, published as U.S. Patent Application Publication No. 2004-0171156 A1 on September 2, 2004 (this content is described herein). Incorporated by reference as a whole in the book).

Aspects of the invention relate to chimeric RNA and bicistronic vectors for modified or mutated CRISPR enzymes (eg Cas9). Chimeric RNAs and bicistronic expression vectors for modified or mutated CRISPR enzymes are preferred. In general, and in particular, in this embodiment the modified or mutated CRISPR enzyme is preferably driven by the CBh promoter. The chimeric RNA can preferably be driven by a Pol III promoter such as the U6 promoter. Ideally, the two are combined. The chimeric guide RNA typically consists of a 20 bp guide sequence (N), which can be ligated into a tracr sequence (extending from the first "U" in the lower strand to the end of the transcript). The tracr sequence can be truncated at various positions as indicated. The guide sequence and the tracr sequence are separated by the tracr mate sequence, and the tracr mate sequence may be GUUUUAGAGCUA. This can be followed by the loop sequence GAAA as shown. Both of these are preferred examples. Applicants have demonstrated Cas9-mediated indels at the human EMX1 and PVALB loci by the SURVEYOR assay. The chiRNA is indicated by its "+ n" symbol, and the crRNA refers to a hybrid RNA in which the guide sequence and the tracr sequence are expressed as separate transcripts. Throughout this application, chimeric RNA can also be referred to as single-guided or synthetic-guided RNA (sgRNA). The loop is preferably GAAA, but is not limited to this sequence, or in fact, only to be 4 bp long. In practice, the preferred loop-forming sequence for use in hairpin structures is 4 nucleotides in length, most preferably the sequence GAAA. However, longer or shorter loop sequences may be used, as may be alternative sequences. The sequence preferably comprises a nucleotide triplet (eg AAA), and additional nucleotides (eg C or G). Examples of loop-forming sequences include CAAA and AAAG. In the practice of any of the methods disclosed herein, without limitation, microinjection, electroperforation, sonoporation, microscopic guns, calcium phosphate mediated transfections, cationic transfections, liposome transfections, dendrimer transfections. , Heat-shock transfection, Nucleofection transfection, Magnetofection, Lipofection, Impalefection, Optical transfection, Incorporated drug-enhanced nucleic acid uptake, and liposomes, immunolipolips, virosomes, or artificial virions. Vectors suitable for cells or embryos can be introduced by one or more methods known in the art, including delivery via transfection. In some methods, the vector is introduced into the embryo by microinjection. One or more vectors can be microinjected into the embryo's nucleus or cytoplasm. In some methods, one or more vectors can be introduced into cells by nucleofection.

The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, polyadenylation signals and transcription termination signals such as polyU sequences). .. For such adjustment elements, see, for example, Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that induce constitutive expression of nucleotide sequences in many types of host cells and those that induce expression of nucleotide sequences only in specific host cells (eg, tissue-specific regulatory sequences). Tissue-specific promoters are primarily expressed in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg liver, pancreas), or specific cell types (eg lymphocytes). Can be derived. Regulatory elements may also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which expression may or may not be tissue- or cell-type specific. In some embodiments, the vector is one or more pol III promoters (eg, 1, 2, 3, 4, 5, or more pol III promoters) and one or more pol II promoters (eg, 1, 2, 3, 4, 5, or more). One, two, three, four, five or more pol II promoters), one or more pol I promoters (eg, 1, 2, 3, 4, 5, or more pol I promoters), Or includes a combination of these. Examples of the pol III promoter include, but are not limited to, the U6 and H1 promoters. Examples of the pol II promoter include, but are not limited to, the retrovirus Raus sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [eg, with the CMV enhancer). See Boshard et al, Cell, 41: 521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. In addition, the term "adjustment element" includes an enhancer element such as WPRE; CMV enhancer; R-U5'segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8 (1), p. 466-472). , 1988); SV40 enhancer; and intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78 (3), p. 1527-31, 1981). Is also included. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to transform, the desired expression level, and the like. The vector can be introduced into a host cell, whereby the transcript, protein, or peptide encoded by the nucleic acid as described herein is a fusion protein or peptide (eg, clustered regular). Shortly spaced Palindrome repeat (CRISPR) transcripts, proteins, enzymes, mutants thereof, fusion proteins thereof, etc.) can be produced. For regulatory sequences, see US Patent Application No. 10 / 491,026, the contents of which are incorporated herein by reference in their entirety. For promoters, reference is made to International Publication No. 2011/028929 and US Patent Application No. 12 / 511,940, the contents of which are incorporated herein by reference in their entirety.

Vectors can be designed for expression of CRISPR transcripts (eg, nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, the CRISPR transcript can be expressed in bacterial cells, such as Escherichia coli, insect cells (using a baculovirus expression vector), yeast cells, or mammalian cells. For suitable host cells, see Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. It is discussed in detail in (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro using, for example, the T7 promoter regulatory sequence and T7 polymerase.

The vector may be introduced into a prokaryote or prokaryotic cell and propagated. In some embodiments, the plasmid is amplified to amplify a copy of the vector to be introduced into the eukaryotic cell or as an intermediate vector in the production of the vector to be introduced into the eukaryotic cell (eg, as part of a viral vector packaging system). ), Prokaryotes are used. In some embodiments, a prokaryote is used to amplify a copy of the vector to express one or more nucleic acids, thereby delivering one or more proteins, eg, to a host cell or host organism. I will provide a. Expression of proteins in prokaryotic organisms is often carried out in Escherichia coli using a vector containing a constitutive or inducible promoter that leads to the expression of either a fusion protein or a non-fusion protein. The fusion vector adds a number of amino acids to the amino terminus of the protein encoded therein, eg, a recombinant protein. Such fusion vectors include (i) increased expression of recombinant protein; (ii) increased solubility of recombinant protein; and (iii) promotion of purification of recombinant protein by acting as a ligand in affinity purification. Can serve more than one purpose. In many cases, the fusion expression vector introduces a proteolytic cleavage site at the junction of the fusion moiety and the recombinant protein so that the recombinant protein can be separated from the fusion moiety after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase. Examples of fusion expression vectors are pGEX (Pharmacia Biolabs Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.), And pALI, Mass. ), Each of which fuses glutathione S-transferase (GST), maltose E-binding protein, or protein A to the target recombinant protein. Examples of suitable inducible non-fused E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studio et al., GENE EXPRESSION TECHNOLOGY: METHODS). IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987.EMBO J.6: 229-234), pMFa (Kuijan and Herskowz). -943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), And picZ (InVictorogen Corp, San Diego, Calif). In some embodiments, the vector uses a baculovirus expression vector to drive protein expression in insect cells. Baculovirus vectors that can be used for protein expression in cultured insect cells (eg, SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series. (Lucklow and Summers, 1989. Virus 170: 31-39).

In some embodiments, the vector has the ability to drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the regulatory function of expression vectors is typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Simian virus 40, and other viruses disclosed herein and known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, eg, Sambrook, et al. , MOLECULAR Cloning: A LABORATORY MANUAL. 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. et al. Y. , 1989, Chapters 16 and 17.

In some embodiments, recombinant mammalian expression vectors are capable of preferentially directing nucleic acid expression in specific cell types (eg, tissue-specific regulatory elements are used for nucleic acid expression). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoters (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphatic system-specific promoters (Calame and Eaton,). 1988. Adv. Immunol. 43: 235-275), specifically T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33). : 729-740; QUEEN and Baltimore, 1983. Cell 33: 741-748) promoters, neurospecific promoters (eg, neurofilament promoters; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473) -5477), pancreatic-specific promoter (Eld, et al., 1985. Science 230: 912-916), and mammary gland-specific promoter (eg, lactation promoter; US Pat. No. 4,873,316, and European Patent Application Publication No. 264,166). Also included are developmentally regulated promoters such as the mouse hox promoter (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tiggman, 1989. Genes Dev. 3: 537-546). Will be done. For these prokaryotic and eukaryotic vectors, see US Pat. No. 6,750,059, the contents of which are incorporated herein by reference in their entirety. Other embodiments of the invention may also relate to the use of viral vectors, including US Patent Application No. 13 / 092,085, the contents of which are incorporated herein by reference in their entirety. .. Tissue-specific regulatory elements are known in the art and include, in this regard, US Pat. No. 7,776,321, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the regulatory elements are operably linked to one or more elements of the CRISPR system to drive the expression of one or more elements of the CRISPR system. CRISPR (clustered, regularly spaced, short-interval palindrome repeats), also commonly known as SPIDR (Spacer Interspersed Direct Repeat), is usually a family of DNA loci specific to a particular bacterial species. To configure. The CRISPR locus is E. coli (Ishino et al., J. Bacteriol., 169: 5249-5433 [1987]; and Nakata et al., J. Bacteriol., 171: 3553-3556 [1989]. ) Includes a characteristic class of scattered short-chain sequence repeats (SSRs) and related genes. Similar interspersed SSRs have been identified in Haloferax medialanei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (Mycobacterium tuberculosis). Microbiol., 10: 1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5: 254-263 [1999]; Massepohl et al., Biochim. Biophyss. Acta 1307: 26-30 [ 1996]; and Mojica et al., Mol. Microbiol., 17: 85-93 [1995]). The CRISPR locus is typically referred to as a short, regularly spaced repeat (SRSR), unlike other SSRs (Janssen et al., OMICS J. Integral Biol., 6: 23-33 [2002]; and Mojica et al., Mol. Microbiol., 36: 244-246 [2000]). In general, this repeat is a short element that resides in a cluster that is regularly spaced by a unique intervening sequence of substantially constant length (Mojica et al., [2000], supra). Although repeat sequences are highly conserved among strains, the number of interspersed repeats and the sequence of spacer regions typically vary from strain to strain (van Embden et al., J. Bacteriol., 182: 2393-2401). [2000]). The CRISPR locus is, but is not limited to, Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula, Halocarcula, Metacucula Genus (Methanococcus), genus Metanosarcina (Methanosarcina), genus Metanopurus (Pyrus), genus Pyrococcus, genus Picrofilus, genus Pyrophilus, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Termus, Bacillus, Bacillus Listeria), Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Fusobacterium, Arca , Neisseria, Nitrosomonas, Desulfovivrio, Geobacter, Myxococcus, Campylobacter, Campylobacter, Campylobacter, Campylobacter , Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Salmonella, Salmonella s), Yersinia, Treponema, and Thermotogae have been identified in more than 40 prokaryotes (eg, Jansen et al.). , Mol. Microbiol. , 43: 1565-1575 [2002]; and Mojica et al. , [2005]).

In some embodiments, the CRISPR enzyme is one or more heterologous protein domains (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more in addition to the CRISPR enzyme, or more. It is part of a fusion protein that contains (more domains). The CRISPR enzyme fusion protein may comprise any additional protein sequence and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repressor activity, transcription release factor activity, histones. Examples include protein domains having one or more of modifying activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicolacetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein. Examples include self-fluorescent proteins including (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). The CRISPR enzyme binds to, or is not limited to, a DNA molecule, a maltose-binding protein (MBP), an S-tag, a Lex A DNA-binding domain (DBD) fusion, a GAL4 DNA-binding domain fusion, and a simple herpesvirus ( It may be fused to a gene sequence encoding a protein or protein fragment that binds to other cell molecules, including the HSV) BP16 protein fusion. Additional domains that may form part of the fusion protein, including the CRISPR enzyme, are in US Patent Application Publication No. 20110059502 (incorporated herein by reference). In some embodiments, a tagged CRISPR enzyme is used to identify the location of the target sequence.

Unless otherwise indicated, the practice of the present invention utilizes prior arts of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA within the scope of the art in the art. .. Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRET PROTOCOLS IN MOLECULAR BIOLOGY (F.M.A.Ess) , Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

Models of Genetic and Epigenetic States Using the methods of the invention, plants, animals or cells that can be used as disease models can be created. As used herein, "disease" refers to a disease, disorder, or sign in a subject. For example, using the methods of the invention, animals or cells containing modifications to one or more disease-related nucleic acid sequences, or plants, animals or plants in which the expression of one or more disease-related nucleic acid sequences is altered. Can produce cells. Such a nucleic acid sequence may encode a disease-related protein sequence or may be a disease-related control sequence. Therefore, it is understood that in embodiments of the invention the plant, subject, patient, organism or cell can be a non-human subject, patient, organism or cell. Therefore, the present invention provides a plant, animal or cell produced by this method, or a progeny thereof. The offspring may be clones of the produced plant or animal, or may result from sexual reproduction by mating with other individuals of the same species to introgress the desired trait into the offspring. The cells may be in vivo or ex vivo in the case of multicellular organisms, especially animals or plants. In the case where the cells are in culture, a cell line can be established if suitable culture conditions are met, and preferably if the cells are suitable for this purpose (eg, stem cells). Bacterial cell lines produced by the present invention are also envisioned. The cell line is also envisioned.

In some methods, disease models can be used to study the effects of mutations on the onset and / or progression of animals or cells and diseases using means commonly used in disease research. .. Alternatively, such disease models are useful for studying the effects of pharmaceutically active compounds on disease.

In some methods, disease models can be used to assess the effectiveness of promising gene therapy strategies. That is, the disease-related gene or polynucleotide can be modified so that the onset and / or progression of the disease is inhibited or alleviated. In particular, the method comprises modifying a disease-related gene or polynucleotide such that altered protein is produced and, as a result, the animal or cell has an altered response. Thus, in some methods, genetically modified animals can be compared to animals that are predisposed to develop the disease so that the effects of gene therapy events can be assessed.

In another embodiment, the invention provides a method of developing a biologically active agent that regulates cell signaling events associated with a disease gene. The method involves contacting a cell with a test compound with a cell comprising a CRISPR enzyme, a guide sequence attached to a tracr mate sequence, and one or more vectors driving expression of one or more of the tracr sequences; and eg, included in the cell. It involves detecting changes in readings that indicate a decrease or increase in cell signaling events associated with mutations in the disease gene.

A cell model or animal model can be constructed in combination with the methods of the invention to screen for changes in cell function. Such a model can be used to study the effect of genomic sequences modified by the CRISPR complex of the present invention on cell function of interest. For example, cell function models can be used to study the effects of modified genomic sequences on intracellular or extracellular signaling. Alternatively, cell function models can be used to study the effects of modified genomic sequences on sensory cognition. In some such models, one or more genomic sequences associated with biochemical signaling pathways in the model are modified.

Several disease models have been specifically studied. They include the de novo autism risk genes CHD8, KATNAL2, and SCN2A; and the symptomatological autism (Angelman syndrome) gene UBE3A. These genes and the resulting autism model are of course preferred, but help clarify the broad applicability of the invention across the gene and the corresponding model.

Changes in the expression of one or more genomic sequences associated with biochemical signaling pathways are assayed for differences in mRNA levels of the corresponding genes between test model cells and control cells when contacted with a candidate drug. Can be determined by Alternatively, differences in the expression of sequences associated with biochemical signaling pathways are determined by detecting differences in the level of the encoded polypeptide or gene product.

To assay changes caused by agents at the level of mRNA transcripts or corresponding polynucleotides, the nucleic acids contained in the sample are first extracted according to standard methods in the art. For example, Sambrook et al. The mRNA can be isolated using a variety of lytic enzymes or chemical solutions according to the procedure shown in (1989), or extracted with a nucleic acid binding resin according to the accompanying instructions provided by the manufacturer. The mRNA contained in the extracted nucleic acid sample is then subjected to amplification procedures or conventional hybridization assays (eg, Northern Blot Analysis) according to methods widely known in the art or based on the methods exemplified herein. Detected.

For the purposes of the present invention, amplification means any method using primers and polymerases capable of replicating the target sequence with reasonable fidelity. Amplification can be performed by natural or recombinant DNA polymerases such as TaqGold ™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. The preferred amplification method is PCR. Specifically, the isolated RNA can be subjected to a reverse transcription assay combined with a quantitative polymerase chain reaction (RT-PCR) to quantify the expression levels of sequences associated with biochemical signaling pathways. ..

Detection of gene expression levels can be done in real time during the amplification assay. In one aspect, the amplification product can be directly visualized with a fluorescent DNA binder, such as, but not limited to, a DNA intercalating agent and a DNA groove binder. Since the amount of intercalating agent incorporated into a double-stranded DNA molecule is typically proportional to the amount of amplified DNA product, it is convenient to intercalate using conventional optical systems in the art. By quantifying the fluorescence of the curate dye, the amount of amplification product can be determined. Suitable DNA-binding dyes for this application include SYBR green, SYBR blue, DAPI, propidium iodine, Housete, SYBR gold, ethidium bromide, acridine, proflavin, acridine orange, acridine flavin, fluorescent coumarin, elliptycin, and the like. Examples thereof include daunomycin, chloroquin, distamicin D, chromomycin, homidium, mitramycin, ruthenium polypyridyl, anthramycin and the like.

In another aspect, other fluorescent labels, such as sequence-specific probes, can be used in the amplification reaction to facilitate detection and quantification of the amplification product. Probe-based quantitative amplification relies on sequence-specific detection of the desired amplification product. This amplification utilizes a fluorescent target-specific probe (eg, TaqMan® probe) that results in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are well established in the art and are taught in US Pat. No. 5,210,015.

In yet another embodiment, conventional hybridization assays can be performed using hybridization probes that share sequence homology with sequences associated with biochemical signaling pathways. Typically, the probe is capable of hybridizing to a stable complex with a sequence associated with a biochemical signaling pathway contained within a biological sample obtained from a subject. It will be appreciated by those skilled in the art that when antisense is used as a probe nucleic acid, the target polynucleotide provided in the sample will be selected to be complementary to the sequence of the antisense nucleic acid. Conversely, if the nucleotide probe is a sense nucleic acid, the target polynucleotide is selected to be complementary to the sequence of the sense nucleic acid.

Hybridization can be performed under conditions of various stringencies. Hybridization conditions suitable for practicing the present invention are those in which the cognitive interaction between the probe and the sequence associated with the biochemical signaling pathway is sufficiently specific and stable. Conditions that increase the stringency of hybridization reactions are widely known and published in the art. See, for example, (Sambrook, et al., (1989); Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, second edition). Hybridization assays can be formed using probes immobilized on any solid support, including, but not limited to, nitrocellulose, glass, silicon, and various gene arrays. Preferred hybridization assays are performed on high density gene chips as described in US Pat. No. 5,445,934.

To conveniently detect the probe-target complex formed during the hybridization assay, the nucleotide probe is conjugated to a detectable label. Detectable labels suitable for use in the present invention include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical or chemical means. A wide variety of suitable detectable labels are known in the art, including fluorescent or chemiluminescent labels, radioisotope labels, enzymes or other ligands. In a preferred embodiment, it would be desirable to use a fluorescent label or enzyme tag such as digoxygenin, β-galactosidase, urease, alkaline phosphatase or peroxidase, avidin / biotin complex.

The detection method used to detect or quantify hybridization intensity will typically depend on the label selected above. For example, radiolabels can be detected using photographic film or a phosphorimager. Fluorescent markers can be detected and quantified using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing the enzyme with a substrate and measuring the reaction products produced by the action of the enzyme on the substrate; and finally, colorimetric labels are simply colored labels. Is detected by visualizing.

Changes in drug-induced sequence expression associated with biochemical signaling pathways can also be determined by examining the corresponding gene product. Determining protein levels typically involves a) contacting a protein contained in a biological sample with an agent that specifically binds to a protein associated with a biochemical signaling pathway; and (b) its. Any drug thus formed: involves the step of identifying a protein complex. In one aspect of this embodiment, the agent that specifically binds to a protein associated with a biochemical signaling pathway is an antibody, preferably a monoclonal antibody.

The reaction is a protein associated with a biochemical signaling pathway obtained from a test sample under conditions that allow the formation of a complex between the drug and the protein associated with the biochemical signaling pathway. It is carried out by bringing the drug into contact with the sample. The formation of the complex can be detected directly or indirectly according to standard procedures in the art. In a direct detection method, the drug is provided with a detectable label and the unreacted drug can be removed from the complex; thus the amount of label remaining indicates the amount of complex formed. For such methods, it is preferable to select a label that remains bound to the drug even under stringent wash conditions. The label preferably does not interfere with the binding reaction. Alternatively, indirect detection procedures may use agents containing labels that have been chemically or enzymatically introduced. The desired label generally does not interfere with the binding or stability of the resulting drug: polypeptide complex. However, the label is typically designed to be accessible to the antibody for effective binding and thus generation of a detectable signal.

A wide variety of labels suitable for detecting protein levels are known in the art. Non-limiting examples include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.

The amount of drug: polypeptide complex formed during the binding reaction can be quantified by standard quantitative assays. As described above, the formation of the drug: polypeptide complex can be measured directly by the amount of label remaining at the binding site. In alternatives, proteins associated with biochemical signaling pathways are tested for their ability to compete with labeled analogs for binding sites on a particular drug. In this competitive assay, the amount of label captured is inversely proportional to the amount of protein sequence associated with the biochemical signaling pathway present in the test sample.

Many protein analysis techniques based on the general principles outlined above are available in the art. This includes, but is not limited to, radioimmunoassay, ELISA (enzyme-linked immunoradiometric assay), "sandwich" immunoassay, immunoradiometric assay, in-situ immunoassay (eg, using colloidal gold, enzyme or radioisotope labeling). , Western blot analysis, immunoprecipitation assay, immunofluorescence assay, and SDS-PAGE.

Antibodies that specifically recognize or bind to proteins associated with biochemical signaling pathways are preferred for performing the aforementioned protein analysis. If desired, antibodies that recognize certain types of post-translational modifications (eg, biochemical signaling pathway-induced modifications) can be used. Post-translational modifications include, but are not limited to, glycosylation, lipidation, acetylation, and phosphorylation. These antibodies may be purchased from commercial suppliers. For example, anti-phosphotyrosine antibodies that specifically recognize tyrosine phosphorylated proteins are available from many suppliers, including Invitrogen and PerkinElmer. Anti-phosphotyrosine antibodies are particularly useful in the detection of proteins that are otherwise phosphorylated at their tyrosine residues in response to ER stress. Such proteins include, but are not limited to, eukaryotic initiation factor 2α (eIF-2α). Alternatively, these antibodies can be made by immunizing a host animal or antibody-producing cell with a target protein that exhibits the desired post-translational modification using conventional polyclonal or monoclonal antibody techniques.

In practicing the subject methods, it may be desirable to identify expression patterns of proteins associated with biochemical signaling pathways in different body tissues, different cell types, and / or different intracellular structures. These tests can be performed using tissue-specific, cell-specific, or intracellular structure-specific antibodies that have the ability to bind protein markers that are preferentially expressed in a particular tissue, cell type, or intracellular structure. it can.

Changes in gene expression associated with biochemical signaling pathways can also be determined by examining changes in gene product activity compared to control cells. Assays for drug-induced changes in protein activity associated with biochemical signaling pathways may depend on the biological activity and / or signaling pathway being investigated. For example, if the protein is a kinase, changes in its ability to phosphorylate one or more downstream substrates can be determined by various assays known in the art. Representative assays include, but are not limited to, immunoblots and immunoprecipitation with antibodies such as anti-phosphotyrosine antibodies that recognize phosphorylated proteins. In addition, kinase activity is measured by high-throughput chemiluminescent assays such as AlphaScreen ™ (available from PerkinElmer) and eTag ™ assay (Chan-Hui, et al. (2003) Clinical Immunology 111: 162-174). Can be detected by.

A pH-sensitive molecule, such as a fluorescent pH dye, can be used as the reporter molecule when the protein associated with the biochemical signaling pathway is part of a signaling cascade that results in variations in intracellular pH conditions. In another example, where the protein associated with the biochemical signaling pathway is an ion channel, fluctuations in membrane potential and / or intracellular ion concentration can be monitored. Many commercial kits and high-throughput devices are particularly suitable for rapid and robust screening for regulators of ion channels. Typical devices include FLIPR ™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences). These instruments are capable of simultaneously detecting reactions in more than 1000 sample wells of a microplate and providing real-time measurements and functional data within 1 second or even 1 millisecond.

In the practice of any of the methods disclosed herein, without limitation, microtransfection, electroporation, sonoporation, microscopic guns, calcium phosphate mediated transfection, cationic transfection, liposomal transfection, dendrimer trait Transfection, heat shock transfection, nucleofection transfection, magnetation, lipofection, impalefection, optical transfection, nucleic acid uptake enhanced by proprietary agents, and liposomes, immunoliposomes, virosomes, or artificial Vectors suitable for cells or embryos can be introduced by one or more methods known in the art, including delivery via virions. In some methods, the vector is introduced into the embryo by microinjection. One or more vectors can be microinjected into the embryo's nucleus or cytoplasm. In some methods, one or more vectors can be introduced into cells by nucleofection.

Target locus, target polynucleotide; The target polynucleotide of the PAM sequence CRISPR complex may be any polynucleotide endogenous or exogenous to eukaryotic cells. For example, the target polynucleotide may be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or junk DNA). The target may be a control element or regulatory element or promoter or enhancer or silencer. The promoter may be in the region of +200 bp or even + 1000 bp from TTS in some embodiments. In some embodiments, the control area may be an enhancer. Enhancers are typically above + 1000 bp from TTS. More specifically, expression of eukaryotic protein-encoding genes is generally regulated through multiple cis-acting transcriptional regulatory regions. Some control elements are located near the initiation site (proximal promoter elements), while others are distant (enhancers and silencers). The promoter determines the transcription initiation site and leads to the binding of RNA polymerase II. Three types of promoter sequences have been identified in eukaryotic DNA. The most common TATA boxes are widely used in genes that are transcribed at high speed. Initiating promoters are rarely found in some genes, and CpG islands are a hallmark of transcribed genes. The promoter proximal element is within approximately 200 base pairs from the initiation site. Several such elements, containing up to about 20 base pairs, can aid in the regulation of specific genes. Enhancers, typically about 100 to 200 base pairs in length, contain multiple 8 bp to 20 bp control elements. They can be located 200 base pairs to tens of kilobases upstream or downstream from the promoter, within the intron range, or downstream from the last exon of the gene. Promoter proximal elements and enhancers may be cell type specific and may function only in specific differentiated cell types. However, any of these regions can be a target sequence and is included in the concept that the target may be a control element or regulatory element or a promoter or enhancer or silencer.

Typically, in the context of an endogenous nucleic acid targeting system, a nucleic acid targeting complex, including a guide RNA that hybridizes to a target sequence and forms a complex with one or more nucleic acid targeting effector proteins, is formed. And in or near the target sequence (eg, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more) Cleavage of one or both DNA or RNA strands occurs. As used herein, the term "one or more sequences associated with a target locus of interest" is used in the vicinity of the target sequence (eg, 1, 2, 3, 4, 5, 6 from the target sequence). Refers to a sequence located in the range of 7, 8, 9, 10, 20, 50 base pairs or more, where the target sequence is contained within the target locus of interest).

Although not desired to be constrained by theory, it is believed that the target sequence must be accompanied by a PAM (protospacer flanking motif), a short sequence recognized by the CRISPR complex. The exact sequence and length requirements of the PAM will vary depending on the CRISPR enzyme used, but the PAM is typically a 2-5 base pair sequence flanking the protospacer (ie, the target sequence). Examples of PAM sequences are shown in the Examples section below, and those skilled in the art will be able to identify additional PAM sequences to be used with a given CRISPR enzyme. In addition, by engineering the PAM Interaction (PI) domain, it is possible to program PAM specificity, improve the fidelity of target site recognition, and increase the versatility of Cas, eg Cas9 genome engineering platforms. Can be. Cas proteins such as the Cas9 protein are described, for example, by Kleinstiber BP et al. "Engineered CRISPR-Cas9 nucleoses with alternate PAM Specificities" with altered PAM specificity. Nature. 2015 Jul 23; 523 (7561): 481-5. It can be engineered to alter its PAM specificity as described in doi: 10.1038 / nature14592. In some embodiments, the method comprises attaching the CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex is: Contains a CRISPR enzyme complexed with a guide sequence that hybridizes to the target sequence within the target polynucleotide, the guide sequence is ligated to the tracr mate sequence, and then the tracr mate sequence hybridizes to the tracr sequence. .. In one aspect, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises the step of binding a CRISPR complex to a polynucleotide that causes an increase or decrease in expression of the polynucleotide; where the CRISPR complex is said to be the poly. It contains a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence within a nucleotide, the guide sequence is linked to a tracr mate sequence, and then the tracr mate sequence hybridizes to the tracr sequence. Similar considerations and conditions apply to methods of modifying the target polynucleotide as described above. In practice, these sampling, culturing and reintroduction options apply to all aspects of the invention. In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal, and modifying one or more cells. Culturing can be done in Exobibo at any stage. The one or more cells may be further reintroduced into a non-human animal or plant. For cells to be reintroduced, it is particularly preferred that the cells are stem cells.

In fact, in any aspect of the invention, the CRISPR complex can include a CRISPR enzyme that forms a complex with a guide sequence that hybridizes with a target sequence, the guide sequence may be linked to a tracr mate sequence. Then the tracr mate sequence can hybridize to the tracr sequence.

The present invention relates to the engineering and optimization of systems, methods and compositions used to control gene expression involving sequence targeting of the CRISPR-Cas system and its components, such as genomic perturbation or gene editing. The Cas enzyme is Cas9. The advantage of this method is that the CRISPR system minimizes or avoids off-target binding and the resulting side effects. This is achieved using a system configured to have a high degree of sequence specificity for the target DNA.

With respect to the CRISPR-Cas complex or system, preferably the tracr sequence has one or more hairpins and is at least 30 nucleotides in length, at least 40 nucleotides in length, or at least 50 nucleotides in length; the guide sequence is 10 to 30 nucleotides in length. Long, the CRISPR / Cas enzyme is a type II Cas9 enzyme.

The target polynucleotides of the CRISPR complex are both entitled "System Methods and Compositions for Sequence Manipulation (SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION)", December 12, 2012 and January 2013, respectively. US Provisional Patent Application No. 61/736 with Broad Reference Nos. BI-2011 / 008 / WSGR Reference No. 44063-701.101 and BI-2011 / 008 / WSGR Reference No. 44063-701.102, respectively, filed on the 2nd. , 527 and 61 / 748,427 (all of which are incorporated herein by reference in their entirety), as well as a number of disease-related genes and polynucleotides. Genes and polynucleotides associated with biochemical signaling pathways can be mentioned.

Examples of target polynucleotides include sequences associated with biochemical signaling pathways, such as genes or polynucleotides associated with biochemical signaling pathways. Examples of target polynucleotides include disease-related genes or polynucleotides. A "disease-related" gene or polynucleotide is any gene that produces a transcription or translation product at an abnormal level or in an abnormal form in cells derived from the affected tissue as compared to a non-disease control tissue or cell. Or refers to a polynucleotide. It may be a gene that becomes expressed at abnormally high levels; it may be a gene that becomes expressed at abnormally low levels, where changes in expression correlate with the development and / or progression of the disease. To do. Disease-related genes also refer to genes with one or more mutations or genetic variations that are directly involved in the cause of the disease or have linkage disequilibrium with one or more genes involved therein. The transcribed or translated product may be known or unknown, and may be at normal or abnormal levels.

Genome Wide Knockout Screening The CRISPR-Cas proteins and systems described herein can be used to perform efficient and cost effective functional genome screening. The CRISPR-Cas genome-wide library can be used for such screening. Such screenings and libraries may be able to determine how gene function, cellular pathways involved in a gene, and any changes in gene expression can result in a particular biological process. The advantage of the present invention is that the CRISPR system avoids off-target binding and the resulting side effects. This is achieved using a system configured to have a high degree of sequence specificity for the target DNA.

Genome-wide libraries include multiple CRISPR-Cas-based guide RNAs, including guide sequences capable of targeting multiple target sequences at multiple genomic loci within a eukaryotic cell population, as described herein. obtain. The cell population may be an embryonic stem (ES) cell population. The target sequence at the genomic locus may be a non-coding sequence. The non-coding sequence can be an intron, a regulatory sequence, a splice site, a 3'UTR, a 5'UTR, or a polyadenylation signal. The targeting can alter the gene function of one or more gene products. Targeting can result in knockout of gene function. Targeting a gene product can include more than one guide RNA. The gene product can be targeted by 2, 3, 4, 5, 6, 7, 8, 9, or 10 guide RNAs, preferably 3-4 per gene. Off-target modifications can be minimized (eg, "Off-target modifications may be minimized (See, eg., DNA)," as incorporated herein by reference, "DNA targeting specificity of RNA-guided Cas9 nuclease. Targeting specialty of RNA-guided Cas9 nucleicase) ”. Hsu, P., Scott, D., Weinstein, J., Ran, FA., Konermann, S., Agawala, V., Li, Y. , Wu, X., Shalem, O., Cradick, TJ., Marraffini, LA., Bao, G., & Zhang, F. Nat Biotechnol doi: 10.1038 / nbt. 2647 (2013). It may be the targeting of 100 or more sequences, the targeting of about 1000 or more sequences, the targeting of about 20,000 or more sequences, the targeting of the entire genome. Nuclease may be the targeting of a panel of target sequences focused on relevant or desirable pathways. The pathway may be the immune pathway. The pathway may be the cell division pathway. May be good.

One aspect of the invention includes a genome-wide library that may include multiple CRISPR-Cas-based guide RNAs that may include guide sequences that can target multiple target sequences at multiple genomic loci, wherein said targeting. Causes knockout of gene function. This library may potentially contain guide RNAs that target individual genes within the organism's genome.

In some embodiments of the invention, the organism or subject is a eukaryote (including mammals including humans) or non-human eukaryotes or non-human or non-human mammals. In some embodiments, the organism or subject is a non-human animal and may be an arthropod, such as an insect, or a nematode. In some methods of the invention, the organism or subject is a plant. In some methods of the invention, the organism or subject is a mammal or non-human mammal. Non-human mammals may be, for example, rodents (preferably mice or rats), ungulates, or primates. In some methods of the invention, the organism or subject is an alga, including microalgae, or a fungus.

For knockout of gene function, I. Cas protein, and II. Steps to introduce a vector system of one or more vectors, including an engineered non-naturally occurring CRISPR-Cas system containing one or more guide RNAs, into each cell in a cell population [Components I and II are of the same system or Steps to integrate components I and II into each cell [may be on different vectors], the guide sequence targets a unique gene within each cell, the Cas protein is operably linked to a regulatory element, A guide RNA containing a guide sequence, when transcribed, induces sequence-specific binding of the CRISPR-Cas system to a target sequence at the genomic locus of a unique gene], inducing cleavage of the genomic locus by the Cas protein. A step and a step of identifying different knockout mutations in multiple unique genes within each cell of the cell population may be included, thereby generating a gene knockout cell library. The present invention includes that the cell population is a eukaryotic cell population and, in a preferred embodiment, the cell population is a population of embryonic stem (ES) cells.

The one or more vectors may be plasmid vectors. The vector may be a single vector to target cells containing Cas9, sgRNA, and optionally a selectable marker. Although not constrained by theory, the ability to simultaneously deliver Cas9 and sgRNA in a single vector allows it to be applied to any cell type of interest without the need to initially create a Cas9 expressing cell line. .. The regulatory element may be an inducible promoter. The inducible promoter may be a doxycycline inducible promoter. In some methods of the invention, the expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase. Confirmation of various knockout mutations can be done by whole exome sequencing. Knockout mutations can be achieved in over 100 unique genes. Knockout mutations can be achieved in over 1000 unique genes. Knockout mutations can be achieved in over 20,000 unique genes. Knockout mutations can be achieved throughout the genome. Knockout of gene function may be achieved in multiple unique genes that function in a particular physiological pathway or condition. The pathway or condition may be an immune pathway or condition. The pathway or condition may be a cell division pathway or condition.

The invention also provides a kit containing the genome-wide library described herein. The kit may include a single container containing a vector or plasmid containing the libraries of the invention. The kit can also include a panel containing a selected portion of a unique CRISPR-Cas-based guide RNA containing guide sequences from the libraries of the invention, the portion selected herein being specific physiology. It shows the target conditions. The present invention includes targeting about 100 or more sequences, about 1000 or more sequences or about 20,000 or more sequences, or the entire genome. In addition, the panel of target sequences may focus on relevant or desirable pathways, such as immune pathways or cell division.

In a further aspect of the invention, the Cas9 enzyme may contain one or more mutations and can be used as a common DNA binding protein with or without fusion to a functional domain. The mutation may be an artificially introduced mutation or a gain-of-function or loss-of-function mutation. Mutations can include, but are not limited to, mutations in one of the catalytic domains (D10 and H840) in the RuvC and HNH catalytic domains, respectively. Further mutations have been characterized. In one aspect of the invention, the functional domain may be a transcription activation domain, which may be VP64. In another aspect of the invention, the functional domain may be a transcriptional repressor domain, which can be KRAB or SID4X. Other aspects of the invention are, but are not limited to, transcriptional activators, repressors, recombinases, transposases, histone remodelers, demethylases, DNA methyltransferases, cryptochromes, photoinducible / regulatory domains or chemical inducible / It relates to a mutant Cas9 enzyme fused to a domain containing a regulatory domain. Some methods of the invention may include the step of inducing the expression of a target gene. In one embodiment, induction of expression by targeting multiple target sequences at multiple genomic loci within a eukaryotic cell population is by use of a functional domain.

Useful in the practice of the present invention, see:
"Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells". "Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells". Shalem, O.D. , Sanjana, NE. , Hartenian, E.I. , Shi, X. , Scott, DA. , Mikkelson, T.M. , Heckl, D.I. , Evert, BL. , Rot, DE. , Doench, JG. , Zhang, F. et al. Science Dec 12. (2013). [Epub ahead of print]; The final revised version was published as: Science. 2014 Jan 3; 343 (6166): 84-87.
-Shalem et al. Revolves around a novel method for exploring gene function on a genome-wide scale. Their study conducted negative and positive selection in human cells by delivering a genome-wide CRISPR-Cas9 knockout (GeCKO) library that targets 18,080 genes with 64,751 unique guide sequences. It was shown that both screenings were possible. First, the authors have shown that the GeCKO library is used to identify genes essential for cell viability in cancer and pluripotent stem cells. The authors then screened genes in the melanoma model whose deficiency was associated with resistance to vemurafenib, a therapeutic agent that inhibits the mutant protein kinase BRAF. Their study revealed that the top-ranked candidates included the already validated genes NF1 and MED12 and the new hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high degree of consistency and high hit confirmation rates among independent guide RNAs targeting the same gene, thus demonstrating the promise of genome-wide screening with Cas9.

See also US Patent Application Publication No. 20140357530; and International Publication No. 2014093701 Pamphlet (incorporated herein by reference herein).

Functional Alteration and Screening In some embodiments, one or more functional domains associate with a CRISPR enzyme, such as a type II Cas9 enzyme.

In some embodiments, one or more functional domains are described, for example, in Konnerman et al. It associates with the adapter protein as used with the modification guide of (Nature 517,583-588,29 January 2015).

In some embodiments, one or more functional domains are associated with dead sgRNA (dRNA). In some embodiments, for example, Dahlman et al. , "Orthogonal gene control with a catalytically active Cas9 nucleose" (printing), a dRNA complex containing active Cas9 is regulated by a functional domain on a gene locus. On the other hand, sgRNA leads to DNA cleavage by active cas9 at another locus. In some embodiments, the dRNA is selected to maximize regulatory selectivity for the locus of interest as compared to off-target regulation. In some embodiments, the dRNA is selected so that target gene regulation is maximized and target cleavage is minimized.

For the purposes of the discussion below, reference to a functional domain can be a functional domain associated with a CRISPR enzyme or a functional domain associated with an adapter protein.

In the practice of the present invention, individual one or more RNA loops or individual one or more RNA loops or individual capable of recruiting an adapter protein capable of binding to individual one or more sequences. Insertion of one or more sequences may extend the loop of the sgRNA without colliding with the Cas9 protein. Adapter proteins can include, but are not limited to, orthogonal RNA-binding protein / aptamer combinations that fall within the diversity of bacteriophage coat proteins. The list of such coated proteins includes, but is not limited to: Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. These adapter proteins or orthogonal RNA-binding proteins can further recruit effector proteins or fusions containing one or more functional domains. In some embodiments, the functional domains are transposase domain, integrase domain, recombinase domain, resolverase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain. , Histone deacetylase domain, nuclease domain, repressor domain, activator domain, transcriptional regulatory protein (or transcription complex recruitment) domain, cell uptake activity related domain, nucleic acid binding domain, antibody presentation domain, histone modifier, histone modification Enzyme recruiters; histone modifying enzyme inhibitors, histone methyltransferases, histone demethylases, histone kinases, histone phosphatases, histone ribosylases, histone deribosyrase, histone ubiquitinase, histone deubiquitinase, histone biotinase and It can be selected from the group consisting of histone tail proteases. In some preferred embodiments, the functional domain is a transcriptional activation domain, eg, without limitation, VP64, p65, MyoD1, HSF1, RTA, SET7 / 9 or histone acetyltransferase. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is a SID, or a concatemer of the SID (eg, SID4X). In some embodiments, the functional domain is an epigenetic modified domain, providing an epigenetic modified enzyme. In some embodiments, the functional domain is the activation domain, which may be the P65 activation domain.

In some embodiments, one or more functional domains are NLS (Nuclear Localization Sequence) or NES (Nuclear Translocation Signal). In some embodiments, the one or more functional domains are transcriptional activation domains, including VP64, p65, MyoD1, HSF1, RTA, SET7 / 9 and histone acetyltransferase. Other references herein to activation (or activator) domains for those associated with CRISPR enzymes include any known transcriptional activation domains and specifically, VP64, p65, MyoD1, HSF1, Includes RTA, SET7 / 9 or histone acetyltransferase.

In some embodiments, the one or more functional domains are transcriptional repressor domains. In some embodiments, the transcriptional repressor domain is the KRAB domain. In some embodiments, the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or SID4X domain.

In some embodiments, the one or more functional domains are methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity. Alternatively, it has one or more activities including nucleic acid binding activity.

Histone modified domains are also preferred in some embodiments. An exemplary histone modification domain is discussed below. Transposase domains, HR (homologous recombination) mechanism domains, recombinase domains, and / or integrase domains are also preferred as the functional domain. In some embodiments, the DNA integration activity comprises an HR mechanism domain, an integrase domain, a recombinase domain and / or a transposase domain. Histone acetyltransferase is preferred in some embodiments.

In some embodiments, the DNA cleaving activity is due to a nuclease. In some embodiments, the nuclease comprises a Fok1 nuclease. "Dimeric CRISPR RNA-guided FokI nucleoses for high-highly special genome editing", Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. et al. Foden, Visual Thepar, Deepak Reyon, Mathew J. et al. Goodwin, Martin J.M. Aryee, J. et al. See Keith Jung Nature Biotechnology 32 (6): 569-77 (2014), which is a dimeric RNA guide capable of recognizing elongated sequences and editing endogenous genes in human cells with high efficiency. Regarding lower FokI nuclease.

In some embodiments, one or more functional domains are added to the CRISPR enzyme such that the functional domain is placed in a spatial arrangement in which the functional domain can function in its attribution function upon binding to the sgRNA and target. Will be done.

In some embodiments, one or more functional domains are adapters such that when the CRISPR enzyme binds to an sgRNA and target, the functional domain is placed in a spatial arrangement in which the functional domain can function with its attribution function. It is added to the protein.

In some embodiments, the invention provides a composition as discussed herein, wherein one or more functional domains are via a linker as discussed herein, optionally via a GlySer linker. Is added to the CRISPR enzyme or adapter protein.

Endogenous transcriptional repression is often mediated by chromatin-modifying enzymes such as histone methyltransferases (HMTs) and deacetylases (HDACs). Inhibitory histone effector domains are known and an exemplary list is provided below. In this exemplary table, small size proteins and functional truncations were prioritized to facilitate efficient viral packaging (eg, by AAV). However, in general, these domains may include HDACs, histone methyltransferases (HMTs), and histone acetyltransferase (HAT) inhibitors, as well as HDAC and HMT recruitment proteins. Functional domains are, in some embodiments, HDAC effector domains, HDAC recruiter effector domains, histone methyltransferase (HMT) effector domains, histone methyltransferase (HMT) recruiter effector domains, or histone acetyltransferase inhibitor effector domains. It may or may include it.

Therefore, the repressor domain of the present invention can be selected from histone methyltransferase (HMT), histone deacetylase (HDAC), histone acetyltransferase (HAT) inhibitors, and HDAC and HMT recruitment proteins.

The HDAC domain may be any of those in the table above: HDAC8, RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, or SIRT6.

In some embodiments, the functional domain may be the HDAC recruiter effector domain. Preferred examples include those in the table below, namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. Although NcoR is exemplified in this example and preferred, it is envisioned that others in this class may also be useful.

In some embodiments, the functional domain may be a methyltransferase (HMT) effector domain. Preferred examples include those in the table below, namely NUE, vSET, EHMT2 / G9A, SUV39H1, dim-5, KYP, SUVR4, SET4, SET1, SETD8, and TgSET8. Although NUE is exemplified in this embodiment, it is assumed that others in this class may also be useful.

In some embodiments, the functional domain may be the histone methyltransferase (HMT) recruiter effector domain. Preferred examples include those in the table below, namely Hp1a, PHF19, and NIPP1.

In some embodiments, the functional domain may be a histone acetyltransferase inhibitor effector domain. Preferred examples include SET / TAF-1β listed in the table below.

It is also preferred to target endogenous (regulatory) control elements (such as enhancers and silencers) in addition to promoters or promoter proximal elements. Thus, the invention can also be used to target endogenous control elements, including enhancers and silencers, in addition to targeting promoters. These control elements may be located upstream and downstream of the transcription initiation site (TSS), starting at 200 bp and 100 kb away from the TSS. Targeting of known regulatory elements can be used to activate or suppress the gene of interest. In some cases, a single regulatory element can affect the transcription of multiple target genes. Therefore, the transcription of multiple genes can be regulated simultaneously using the targeting of a single regulatory element.

On the other hand, targeting of the putative control element (eg, by tiling the region of the putative control element and around 200 bp to 100 kB of the element) is a means of confirming such element (by measuring transcription of the gene of interest) or novel. It can be used as a means for detecting a control element (for example, by tiling 100 kb upstream and downstream of the TSS of the gene of interest). In addition, targeting putative control elements can be useful in the context of elucidating the genetic causes of disease. Many mutations and common SNP variants associated with disease phenotypes are located outside the coding region. Targeting such regions by either the activation system or the inhibitory system described herein is then followed by a) a set of putative targets (eg, a set of genes located in close proximity to the control element). Or b) Transcriptional readings of either RNAseq or full transcriptome readings by microarrays can follow. This may enable identification of candidate genes that are expected to be involved in disease phenotype. Such candidate genes may be useful as new drug targets.

Histone acetyltransferase (HAT) inhibitors are referred to herein. However, alternatives in some embodiments are those in which one or more functional domains comprise an acetyltransferase, preferably histone acetyltransferase. These are useful in the field of epigenomics, for example, in epigenome search methods. Methods for searching for epigenomes can include, for example, targeting epigenome sequences. Targeting the epigenome sequence can include directing the guide to the epigenome target sequence. Epigenome target sequences may include promoter, silencer or enhancer sequences in some embodiments.

Targeting epigenome sequences by using a CRISPR-Cas enzyme, preferably dead Cas, more preferably a functional domain linked to dead Cas9, as described herein activates or suppresses a promoter, silencer or enhancer. Can be used to

Examples of acetyltransferases are known, but in some embodiments histone acetyltransferases may be included. In some embodiments, histone acetyltransferases may comprise a catalytic core of human acetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6th April 2015).

In some preferred embodiments, the functional domain is linked to the dead Cas9 enzyme to target and activate epigenome sequences such as promoters or enhancers. One or more guides directed to such promoters or enhancers may also be provided to guide the binding of the CRISPR enzyme to such promoters or enhancers.

The term "associating with" is used herein with respect to the association of a functional domain with a CRISPR enzyme or adapter protein. It is used, for example, with respect to how one molecule is "associating" with another molecule, between the adapter protein and the functional domain, or between the CRISPR enzyme and the functional domain. In the case of such protein-protein interactions, this association may be considered in terms of recognition in the way antibodies recognize epitopes. Alternatively, one protein may be associated with another protein via a fusion of the two, for example one subunit may be fused to another. Fusion typically occurs by adding one amino acid sequence to the other amino acid sequence, eg, by splicing the nucleotide sequences encoding each protein or subunit together. Alternatively, it may be considered essentially as a bond or direct link between two molecules, such as a fusion protein. In any case, the fusion protein may contain a linker between the two subunits of interest (ie, between the enzyme and the functional domain or between the adapter protein and the functional domain). Thus, in some embodiments, the CRISPR enzyme or adapter protein is associated with it by binding to a functional domain. In other embodiments, the CRISPR enzyme or adapter protein is associated with the functional domain because the two are optionally fused together via an intermediate linker. Examples of linkers include the GlySer linkers discussed herein.

Binding of functional domains or fusion proteins can be mediated by linkers such as mobile glycine-serine (GlyGlyGlySer) or (GGGS) ₃ or strong α-helix linkers such as (Ala (GluAlaAlaAlaLys) Ala). In this specification, a linker such as (GGGGS) ₃ is preferably used to separate protein or peptide domains. (GGGGS) ₃ is preferred because it is a relatively long linker (15 amino acids). Glycine residues are the most mobile, and serine residues increase the likelihood that the linker is outside the protein. (GGGGS) ₆ (GGGGS) ₉ or (GGGGS) ₁₂ can preferably be used as an alternative. Other preferred alternatives are (GGGGS) ₁ , (GGGGSS) ₂ , (GGGGGS) ₄ , (GGGGGS) ₅ , (GGGGGS) ₇ , (GGGGSS) ₈ , (GGGGGS) ₁₀ , or (GGGGGS) ₁₁ . Alternative linkers are available, but we believe that highly mobile linkers work best to maximize the chances that the two parts of Cas9 will come together and thus Cas9 activity be restored. Be done. One alternative is that the NLS of nucleoplasmin can be used as a linker. For example, a linker can also be used between Cas9 and any functional domain. Again, the (GGGGS) ₃ linker (or its 6, 9, or 12 repeat version) may be used here, or the NLS of nucleoplasmin between Cas9 and the functional domain may be used as the linker. Can be done.

Saturation Mutagenesis In general, with respect to the use of the CRISPR-Cas system, documents including patent applications, patents, and patent publications cited throughout this disclosure use embodiments of the invention as in those documents. Listed with the gain. One or more CRISPR-Cas systems represent cells to determine, for example, important minimal features and individual vulnerabilities of functional elements required for gene expression, drug resistance, and disease improvement. It can be used to perform saturation or deep scanning mutagenesis at genomic loci in combination with the type. Saturation or deep scanning mutagenesis means that any or essentially any DNA base is cleaved within a genomic locus. A library of CRISPR-Cas guide RNAs is introduced into the cell population. This library can be introduced such that each cell receives a single guide RNA (sgRNA). As described herein, low multiplicity of infection (MOI) is used when the library is introduced by transduction of a viral vector. The library may contain sgRNAs that target all sequences upstream of the (protospacer flanking motif) (PAM) sequence at the genomic locus. The library may contain at least 100 non-overlapping genomic sequences upstream of the PAM sequence for every 1000 base pairs within a genomic locus. The library may contain sgRNAs that target sequences upstream of at least one different PAM sequence. One or more CRISPR-Cas systems can contain more than one Cas protein. Any Cas protein as described herein can be used, including orthologs or engineered Cas proteins that recognize different PAM sequences. The frequency of off-target sites for sgRNA can be less than 500. An off-target score can be created to select the lowest off-target site sgRNA. By using an sgRNA that targets the same site in a single experiment, any phenotype determined to be associated with cleavage at the sgRNA target site can be identified. Verification of the target site may also be performed using nickase Cas9 as described herein and two sgRNAs targeting the genomic site of interest. Although not constrained by theory, the target site is a true hit when phenotypic changes are observed in validation experiments.

The genomic locus may contain at least one continuous genomic region. The at least one continuous genomic region can include the entire genome. At least one continuous genomic region may contain functional elements of the genome. The functional element may be within the non-coding region, coding gene, intron region, promoter, or enhancer. The at least one continuous genomic region may contain at least 1 kb, preferably at least 50 kb of genomic DNA. At least one continuous genomic region may contain a transcription factor binding site. At least one continuous genomic region may include a DNase I hypersensitive region. At least one continuous genomic region may include a transcription enhancer or repressor element. At least one continuous genomic region can include sites enriched with epigenetic signatures. At least one continuous genomic DNA region may include an epigenetic insulator. At least one contiguous genomic region can include two or more contiguous genomic regions that physically interact with each other. The interacting genomic regions can be determined by "4C technology". According to 4C technology, Zhao et al. ((2006) Nat Genet 38,1341-7) and US Pat. No. 8,642,295 (both incorporated herein by reference in their entirety) with selected DNA fragments and physical. It is possible to screen the entire genome in an unbiased manner with respect to the DNA segments that interact with. Epigenetic signatures can be histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, DNA methylation, or their lack.

One or more CRISPR-Cas systems can be used in a cell population for saturation or deep scanning mutagenesis. One or more CRISPR-Cas systems can be used in eukaryotic cells, including but not limited to mammalian and plant cells. The cell population may be prokaryotic cells. The eukaryotic cell population may be a population of embryonic stem (ES) cells, nerve cells, epithelial cells, immune cells, endocrine cells, muscle cells, erythrocytes, lymphocytes, plant cells, or yeast cells.

In one aspect, the invention provides a method of screening for functional elements associated with phenotypic changes. The library can be introduced into a cell population adapted to contain the Cas protein. Cells can be classified into at least two groups based on phenotype. The phenotype may be gene expression, cell growth, or cell viability. The relative representation of the guide RNA present in each group is determined, thereby determining the genomic site associated with the phenotypic change by the representation of the guide RNA present in each group. The phenotypic change may be a change in the expression of the gene of interest. The gene of interest can be down-regulated, down-regulated, or knocked out. Cells can be classified into high expression groups and low expression groups. The cell population may include a reporter construct used to determine the phenotype. The reporter construct may include a detectable marker. Cells can be classified by using detectable markers.

In another aspect, the invention provides a method of screening genomic sites associated with chemical compound resistance. The chemical compound can be a drug or a pesticide. The library can be introduced into a cell population adapted to contain the Cas protein, where each cell in the population contains no more than one guide RNA; the cell population is treated with a chemical compound; and chemically. After treatment with the compound, the representation of the guide RNA at a later time point compared to the earlier time point is determined, thereby determining the genomic site associated with chemical compound resistance by enrichment of the guide RNA. The expression of sgRNA may be determined by a deep sequencing method.

A paper entitled "BCL11A Enhancer Dissection by Cas9-mediated in Saturating Mutagenesis", Cumber, M. et al., Useful in the practice of the present invention. C. , Smith, E.I. C. , Sher, F.I. , Pinello, L. et al. , Sanjana, N.M. E. , Salem, O.D. , Chen, D. et al. D. , Schupp, P. et al. G. , Vinjamur, D.I. S. , Garcia, S.A. P. , Luc, S.A. , Kurita, R.M. , Nakamura, Y. et al. , Fujiwara, Y. et al. , Maeda, T.M. , Yuan, G.M. , Zhang, F. et al. , Orkin, S.A. H. , & Bauer, D. E. DOI: 10.1038 / nature15521, published online, September 16, 2015, which is incorporated herein by reference and briefly discussed below:
・ Canver et al. CRISPR-A novel pool for performing insitu saturation mutagenesis of human and mouse BCL11A erythrocyte enhancers associated with fetal hemoglobin (HbF) levels and whose mouse orthologs have already been identified as enhancers required for erythrocyte BCL11A expression. Describes the Cas9 guide RNA library. This approach reveals important minimal features and individual vulnerabilities of these enhancers. Using primary human progenitor cell editing and mouse transgenesis, the authors demonstrated the BCL11A erythrocyte enhancer as a target for HbF reinduction. The authors have created a detailed enhancer map that provides information about therapeutic genome editing.

Methods of Modifying Cells or Organisms Using the CRISPR-Cas System The present invention in some embodiments includes methods of modifying cells or organisms. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. Mammalian cells may be non-human primates, bovine, porcine, rodent or mouse cells. The cells may be non-mammalian eukaryotic cells such as poultry, fish or shrimp. The cell may also be a plant cell. The plant cell may be a crop plant such as cassava, corn, sorghum, wheat, or rice. Plant cells may also be algae, trees or vegetables. Modifications introduced into cells by the present invention can be such as altering cells and cell progeny to enhance the production of biological products such as antibodies, starches, alcohols or other desired cell products. The modifications introduced into a cell by the present invention can be such that changes that alter the biological product produced are included in the cell and its offspring.

The system may contain one or more different vectors. In certain aspects of the invention, the Cas protein is codon-optimized for expression in the desired cell type, preferentially eukaryotic cells, preferably mammalian or human cells.

CRISPR systems can be used in plants One or more CRISPR-Cas systems (eg, single or multiplexed) can be used in conjunction with recent advances in crop genomics. Efficient and cost-effective exploration or editing or manipulation of plant genes or genomes using such one or more CRISPR-Cas systems-eg, rapid exploration and / or selection of plant genes or genomes. And / or for exploration and / or comparison and / or manipulation and / or transformation; for example, creating, identifying, and developing one or more traits or one or more traits. , Optimized, or donated to one or more plants, or transformed the plant genome. Thus, there may be new plants with improved plant production, new combinations of traits or properties, or new plants with enhanced traits. Such one or more CRISPR-Cas systems can be used with respect to plants in site-specific integration (SDI) or gene editing (GE) or any quasi-reverse breeding (NRB) or reverse breeding (RB) technique. .. Regarding the use of the CRISPR-Cas system in plants, the University of Arizona website "CRISPR-PLANT" (http://www.genome.arizona.edu/crispr/) (sponsored by Penn State and AGI) Be done. Embodiments of the present invention can be used in genome editing in plants, or where RNAi or similar genome editing techniques have already been used; for example, Nekrasov, "Easy Plant Genome Editing: CRISPR. / Model using Cas system and target mutation induction in crop plants (Plant genome editing made ease: targeted genome editing in model and crop plants CRISPR / Cas system / Cas system: 3, Plant. 1186/1746-4811-9-39); Brooks, "Efficient genome editing in tomato in the first generation using the CRISPR / Cas9 system" CRISPR / Cas9 system. Plant Physiologic September 2014 pp 114.247757; Shan, "Targeted genome modification of crop plants using the CRISPR-Cas system (Targeted genome editing of group68 sequation of Crop plant68s. ); Feng, "Efficient genome editing in plants using the CRISPR / Cas system", Cell Research (2013) 23: 1229-1232. doi: 10.1038 / cr. 2013.114; Online Publication 20 August 2013; Xie, "RNA-guided genome editing in plants using a CRISPR-Cas system", Mol Plant. 2013 Nov; 6 (6): 1975-83. doi: 10.1093 / mp / sst119. Epub 2013 Aug 17; Xu, "Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas," CRISPR-Cas, "Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas 2014), Zhou et al. , "Use of SNPs for both allele CRISPR mutations in diplomatic mating of the Kimoto perennial plant Populus reveals 4-kumalic acid: CoA ligase specificity and redundancy. in the outcrossing woody perennial Populus revolutions 4-commutation: CoA ligase speciality and CRISPR) ”, New Phytologist (2015) available online; available online (2015) (Forum) 1-4 Target DNA degradation using a CRISPR device that stably supports the genome (Mutated DNA degradation using a CRISPR device carry carried in the host genome) ”, NATURE COMMUNICATIONS 6: 6989. nature. com / genomecommunications DOI: 10.1038 / ncomms7989; US Pat. No. 6,603,061-Agrobacterium-Mediated Plant Transformation Method; US Pat. No. 7,868,14. No.-Plant Genome Sequences and Uses Thereof and U.S. Patent Application Publication No. 2009/0100536-Transgenic Plants with Agrobacterium (Transgenic Plants with Agrobacterium) The entire content and disclosure of each of these is incorporated herein by reference in their entirety). In the practice of the present invention, Morrell et al, "Crop genomics: advances and applications", Nat Rev Genet. 2011 Dec 29; 13 (2): 85-96 Content and Disclosure; each of which is incorporated herein by reference, including with respect to how embodiments herein can be used with respect to plants. .. Thus, references to animal cells herein may also be modified as appropriate and applied to plant cells unless otherwise apparent; and the enzymes and such enzymes herein that have reduced off-target effects are utilized. The system can be used for plant applications, including those described herein.

Sugano et al. (Plant Cell Physiol. 2014 Mar; 55 (3): 475-81. Doi: 10.1093 / pcp / pcu014. Epub 2014 Jan 18) is a liverwort that has emerged as a model species for the study of land plant evolution. Marcantia polymorpha L. We report the application of CRISPR / Cas9 to target mutagenesis in (Marchantia polymorpha L.). M. The U6 promoter of M. polymorpha was identified and cloned to express gRNA. The target sequence of gRNA is M.I. It was designed to disrupt the gene encoding auxin response factor 1 (ARF1) of polymorpha (M. polymorpha). Sugano et al. Using Agrobacterium-mediated transformation with M. tumef. Stable mutants of M. polymorpha in the gametophyte generation were isolated. CRISPR / Cas9-based in vivo site-specific mutagenesis is performed by cauliflower mosaic virus 35S or M. coli. It was achieved by expressing Cas9 using one of the promoters of M. polymorpha EF1α. The isolated mutant individuals showing the auxin-resistant phenotype were not chimeric. In addition, asexual reproduction of T1 plants produced stable mutants. Multiple arf1 alleles were readily constructed using CRISPR / Cas9-based targeted mutagenesis. Sugano et al. Method can be applied to the CRISPR Cas system of the present invention.

Kabaddi et al. (Nucleic Acids Res. 2014 Oct 29; 42 (19): e147. Doi: 10.1093 / nar / gku749. Epub 2014 Aug 13) is an independent RNA polymerase III incorporated into the vector by a convenient golden gate cloning method. We have developed a single lentiviral system that expresses Cas9 variants, reporter genes and up to 4 sgRNAs from the promoter. Each sgRNA is efficiently expressed and can mediate multiple gene editing and sustained transcriptional activation in immortalized and primary human cells. Kabaddi et al. Method can be applied to the CRISPR Cas system of the present invention.

Ling et al. (BMC Plant Biology 2014, 14: 327) has developed a CRISPR / Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as gRNA. This toolkit efficiently produces final constructs with corn codon-optimized Cas9 and one or more gRNAs in just one cloning step, without the need for restriction enzymes other than BsaI. This toolkit has been demonstrated using maize protoplasts, transgenic maize strains, and transgenic Arabidopsis strains and has been shown to exhibit high efficiency and specificity. More importantly, the toolkit was used to detect targeted mutations in three Arabidopsis genes in transgenic seedlings of the T1 generation. Furthermore, mutations in multiple genes could be passed on to the next generation. A (guide RNA) module vector set as a toolkit for multiple genome editing in plants. Lin et al. Toolbox can be applied to the CRISPR Cas system of the present invention.

A protocol for targeting plant genome editing with CRISPR / Cas9 is also available in Volume 1284 of the series Methods in Molecular Biology pp 239-255 10 February 2015. Detailed design, construction, and evaluation of dual gRNAs for plant codon-optimized Cas9 (pcoCas9) -mediated genome editing using Arabidopsis thaliana and Nicotiana benthamiana protoplasts model cell lines. The procedure is involved. Strategies for applying the CRISPR / Cas9 system to the generation of targeted genomic modifications in all plants are also considered. The protocols in this chapter can be applied to the CRISPR Cas system of the present invention.

Ma et al. (Mol Plant. 2015 Aug 3; 8 (8): 1274-84. Doi: 10.016 / j.molp. 2015.04.007) is a convenient and highly efficient multiple genome editing in monocotyledonous and dicotyledonous plants. We report a robust CRISPR / Cas9 vector system utilizing the plant cotyledon-optimized Cas9 gene for. Ma et al. Designed a PCR-based procedure for rapidly creating multiple sgRNA expression cassettes that can be assembled into a binary CRISPR / Cas9 vector with a single round of cloning by golden gate ligation or Gibson assembly. In this system, Ma et al. Edited 46 target sites in rice, with an average mutation rate of 85.4%, most of which were biallelic and homozygous. Ma et al. By simultaneously targeting multiple (up to 8) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites within a single gene, T0 rice and T1 arabidopsis Arabidopsis) provides an example of a loss-of-function gene mutation in a plant. Ma et al. Method can be applied to the CRISPR Cas system of the present invention.

Lower et al. (Plant Physiol. 2015 Aug 21. Pii: pp. 00636.6.2015) also allows multiple genome editing and transcriptional regulation of expressed, silencing or non-coding genes in plants CRISPR / Cas9 toolbox Was developed. This toolbox provides researchers with protocols and reagents for rapidly and efficiently assembling functional CRISPR / Cas9 T-DNA constructs for monocotyledons and dicotyledons using golden gate and gateway cloning methods. To do. It has a complete set of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. T-DNA-based transformation techniques are fundamental to modern plant biotechnology, genetics, molecular biology and physiology. Therefore, Applicants have developed a method for assembling Cas9 (WT, nickase or dCas9) and one or more gRNAs into the desired destination T-DNA vector. This assembly method is based on both golden gate assembly and multisite gateway recombination. The assembly requires three modules. The first module is the Cas9 entry vector, which contains the promoterless Cas9 or its inducing gene adjacent to the attL1 and attR5 sites. The second module is a gRNA entry vector, which contains an entry gRNA expression cassette with adjacent attL5 and attL2 sites. The third module contains an attR1-attR2-containing destination T-DNA vector that provides a promoter of choice for Cas9 expression. Lower et al. Toolbox can be applied to the CRISPR Cas9 system of the present invention.

Petersen ("Towards prizely glycol engineered plants", Plant Biotech Denmal meeting 2015, post-modification of protein production, Copenhagen, Denmark) is desired. We have developed a method of engineering genomic changes in the genus Arabidopsis using CRISPR / Cas9, for example, a method of sugar chain engineering of the genus Arabidopsis. Hebelstrup et al. (Front Plant Sci. 2015 Apr 23; 6: 247) produces crops that express starch-modifying enzymes and that produce products that are usually produced by industrial chemical and / or physical treatment of starch. It outlines starch bioengineering in the provided implanters. The Petersen and Hebelstrup methods can be applied to the CRISPR-Cas9 system of the present invention.

In an advantageous embodiment, the plant may be a tree. The present invention can also utilize the CRISPR Cas system disclosed herein as a herbaceous system (eg, Belhaj et al., Plant Methods 9:39 and Harrison et al., Genes & Development 28: 1859-1872. See). In a particularly advantageous embodiment, the CRISPR Cas system of the present invention can target single nucleotide polymorphisms (SNPs) in trees (eg, Zhou et al., New Phytologist, Volume 208, Issues 2, pages 298-301, See October 2015). Zhou et al. In this study, the authors used the 4-coumarate: CoA ligase (4CL) gene family as a case study to apply the CRISPR Cas system in the Perennial Kimoto Populus to target two 4CL genes. Achieving 100% mutation efficiency, all transformants examined here had biallelic modifications. Zhou et al. In this study, the CRISPR / Cas9 system was extremely sensitive to single nucleotide polymorphisms (SNPs), and cleavage of the third 4CL gene was abolished due to SNPs in the target sequence.

Zhou et al. The method of (New Phytologist, Volume 208, Issue 2, pages 298-301, October 2015) can be applied to the present invention as follows. Two 4CL genes, 4CL1 and 4CL2, respectively associated with lignin and flavonoid biosynthesis, are targeted for CRISPR / Cas9 editing. Populus tremula x alba clone 717-1B4, which is routinely used for transformation, is a branch from genome-sequencing Populus trichocarpa. Therefore, by examining 4CL1 and 4CL2 gRNAs designed from the reference genome with in-house 717 RNA-Seq data, we confirm that there are no SNPs that can limit Cas efficiency. Also included is a third gRNA that is a genomic duplicate of 4L1 designed for 4CL5. The corresponding 717 sequences have one SNP near / inside the PAM of each allele, both of which appear to invalidate targeting by 4CL5-gRNA. All three gRNA target sites are located within the first exon. For 717 transformations, gRNA from the Medicago U6.6 promoter is expressed in a binary vector with codon-optimized human Cas under the control of the CaMV 35S promoter. Transformation with a Cas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2 strains are subjected to amplicon sequencing. The data are then processed to confirm biallelic mutations in all cases.

In plants, pathogens are often host-specific. For example, Fusarium oxysporum f.sp.lycopersici causes tomato wilt, but attacks only tomatoes, F. oxysporum f. Oxysporum f. Dianthii and Puccinia graminis f. Sp. Tritici attack only wheat. Plants have existing induced defenses and resist many pathogens. Mutation and recombination events between plant generations result in susceptibility to genetic variation, especially as pathogens replicate more frequently than plants. Plants may also have non-host resistance, for example, the host and pathogen are incompatible. Also, horizontal resistance, eg, partial resistance to any pathogen strain, typically regulated by many genes, and vertical resistance, eg, other, typically regulated by several genes. There may also be complete resistance to some pathogen strains that are not present to the strain. At the gene-to-gene level, plants and pathogens evolve together, with genetic changes in one equilibrating with changes in the other. Therefore, breeders use natural variability to multiply the most useful genes for yield, quality, homogeneity, cold hardiness and resistance. Sources of resistance genes include natural or exotic varieties, native varieties, relatives of wild plants, and induced mutations (eg, treatment of plant material with mutagens). Using the present invention provides plant breeders with a novel means of inducing mutations. Therefore, those skilled in the art will analyze the genome of the source of the resistance gene and use the present invention in varieties having the desired characteristics or traits to produce the resistance gene with higher accuracy than conventional mutagens. Can be induced, and thus the plant breeding program can be accelerated and improved.

Application to plants and yeast; Application to biofuels Application of Cas9-CRISPR system to plants and yeast Definition:
In general, the term "plant" is any variety of photosynthetic organisms, eukaryotes, of the plant kingdom (Plantae) that grow characteristically by cell division, contain chloroplasts, and have a cell wall composed of cellulose. Concerning unicellular or multicellular organisms. The term plant includes monocotyledonous and dicotyledonous plants. Specifically, the plants include, without limitation, acacia, alfalfa, amaranth, apple, apricot, corn, corn tree, asparagus, avocado, banana, wheat, legume, sugar beet, kabanoki, bunanoki, black strawberry, etc. Blueberries, broccoli, mecabbets, cabbage, canola, cantaloupe, carrots, cassaba, cauliflower, Himalayan cereals, grains, celery, chestnuts, cherry, sugar beet, citrus, clementine, clover, coffee, corn, cotton, sardines, cucumber, itosugi, Eggplant, Nile, Endive, Eucalyptus, Wheat, Fig, Fir, Geranium, Grape, Grapefruit, Lacquer, Hozuki, Gum hemlock, Hickory, Kale, Wheat Fruit, Kohlrabi, Karamatsu, Lettuce, Nira, Lemon, Lime , Fake acacia, pine, maiden hair, corn, mango, maple, melon, millet, mushroom, mustard, cereal, oak, oat wheat, oil palm, okra, onion, orange, ornamental plant or decorative flower or ornamental tree, papaya, palm , Parsley, parsnip, pea, peach, peanut, corn, peat, pepper, oyster, corn, pine, pineapple, corn, corn, pomegranate, potato, pumpkin, red chicory, radish, rapeseed, strawberry, rice, lime , Morokoshi, Benibana, Salyanagi, Soybean, Spinach, Ezomatsu, Pumpkin, Strawberry, Sugar beet, Sugar beet, Sunflower, Sweet corn, Sweet corn, Tangerine, Tea, Tobacco, Tomato, Tree, Wheat, Turbaceous grass, Cub, Vine plant, Walnut , Dutch beet, watermelon, wheat, yamuimo, ichii, and zucchini are intended to include anthropogenic and nude offspring. The term plants also include algae, which are mostly photoautotrophs, largely unified by their lack of roots, leaves and other organs that characterize higher plants.

By using a genome editing method using the CRISPR / Cas9 system as described herein, essentially any plant can be imparted with the desired trait. A wide variety of plants and plant cell lines can be engineered using the nucleic acid constructs of the present disclosure and the various transformation methods described above for the desired physiological and agronomic properties described herein. In a preferred embodiment, the plants and plant cells targeted for engineering are, but are not limited to, cereal crops (eg wheat, corn, rice, millet, barley), fruit crops (eg tomatoes, apples, dicotyledons, etc.). Strawberries, oranges), forage crops (eg alfalfa), root vegetable crops (eg carrots, potatoes, sugar corn, yamuimo), leafy plants (eg lettuce, spinach); flowering plants (eg petunia, roses, kiku) ), Coniferous and pine trees (eg fir, pine); plants used in phytotremation (eg heavy metal storage plants); oil crops (eg sunflowers, rapeseed) and plants used for experimental purposes (eg arabidopsis) Examples include monocotyledonous and dicotyledonous plants such as crops including the genus (Arabidopsis). Thus, the method and the CRISPR-Cas system span a wide range of plants, including, for example, Magnioles, Illiciales, Laurales, Piperales, Aristochiales, Waterlily (Aristochiales). Nymphaeales, Ranunculares, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Tochumeis , Beech, Fagales, Casualinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Biwamodokies, Dilenes Eyes (Malvales), Iraqi (Urticales), Sagaribana (Lecythidales), Violes, Yanagi (Salicales), Fuchsaw (Capparales), Tsuji (Ericales), Iwaume (Ericales), Iwaume (Ericales) Ebenales, Primulales, Rosales, Fabales, Podostemales, Halloragales, Hyoragales, Myrtales, Myrtales, , Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Ruhamnales, Sapindales, Sapindales, Orders Polygales, Umbellales, Genitales, Polymoniales, Lamiales, Pl It can be used in dicotyledonous plants belonging to Pandanales, Pandanales, Pandanales, Pandanales, Rubiales, Dipscales, and Asterales; this method and CR The Cas system includes Pandanales (Alismatales), Pandanales (Hydrocharitales), Ibaramo (Najadales), Pandanales (Triuridales), Pandanales (Commerlinales), Pandanales (Eriocales), Pandanales (Eriocales) ), Juncales, Cyperales, Cyphales, Bromeliales, Zingivales, Areces, Pandanales, Pandanales, Pandanales, Pandanales, Pandanales, Pandanales, Pandanales, Pandanales, Pandanales, Pandanales, Monocotyledonous plants such as those belonging to the order Pandanales (Arales), lilies, and Orchid ales, or plants belonging to the dicotyledonous plants (Gymnospermae), such as the order Pinales, Ginkgo (Pinales) It can be used in those belonging to the order Gingcoales, Pandanales, Pandanales, Cupressales and Gnetales.

The CRISPR / Cas9 lineages and methods of use described herein can be used for a wide range of plant species included in the non-limiting list of genera of dicotyledonous, monozygotic or nude plants below: wolf. The genus Atropa, the genus Alseodaphne, the genus Anacardium, the genus Arachis, the genus Beilschmiedia, the genus Brassica, the genus Brassica, the genus Benibana, the genus Carth Croton, Cucumis, Citrus, Waterlus, Capsicum, Catharanthus, Cocos, Coffea, Coffea (Cucurbita), carrot (Daucus), duguetia (Duguetia), genus Eschcholzia, genus Figus, genus Dutch strawberry (Fragaria), genus Tsunogeshi (Glaucium), genus Soybean (Gly) Gossypium, Helianthus, Hevea, Hyoscymus, Lactuca, Landolphia, Landolphia, Linum, Linum, Linum, Linum ), Lupinus, Manihot, Majorana, Malus, Medicago, Nicotiana, Olivea, Parthenium, Parthenium Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Sakura, Raphanus, Tougoma (Ricinus), Kion (Senecio), Tsuzurafuji (Sinomenium), Hasunohakazura (Stephania), Shirogarashi (Sinapis), Nas (Solanum), Cacao (Theobrom) a), Ryegrass, Trifolium, Trigonella, Vicia, Vinca, Vilis, and Vigna; and Allium, Oryza (Allium), Usyxa (A), Ryegrass, Ryegrass, and Ryegrass. Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festrium, Festrium (Heterocallis), Corn genus (Hordeum), Aokikusa genus (Lemna), Ryegrass genus (Lolium), Basho genus (Musa), Oryza genus (Oryza), Kibi genus (Panicum), Chikarashiba genus (Pannesetum) ), Meadow-grass (Poa), Limegi (Secale), Morokoshi (Sorghum), Wheat (Triticum), Corn (Zea), Fir (Abies), Koyozan (Cunninghamia), Maou (Ephedra) , Corn genus (Picea), pine genus (Pinus), and Togasawara genus (Pseudotsuga).

The CRISPR / Cas9 system and usage also include, for example, Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta. ) (Cystal algae), several eukaryotic phyla, including Eustigmatophyta and whirlpool algae, as well as prokaryotic phyla, Cyanobacteria (Algae) It can also be used for a wide range of "algae" or "algae cells", including algae. The term "algae" includes, for example, the genus Amphora, the genus Anabaena, the genus Anxtrodesmis, the genus Botriococcus, the genus Chaetoceros, the genus Chaetoceros, the genus Chlamidolamonas. Chlorella, Chlorococcum, Cycrotella, Cylindroteca, Dunaliella, Emiliania, Emiliana, Euglena, Euglena, Euglena, Euglena, Euglena, Euglena Genus (Isochrysis), genus Monocrisis (Monochrysis), genus Monorafidium, genus Nannochloris, genus Nannochloropsis, genus Nannochlopsis, genus Funagatakeiso, genus Navichlo Genus (Nephroselmis), genus Nitzsia, genus Nodularia, genus Nostock, genus Ochromonas, genus Ocystics, genus Ocystis, genus Oscillatora, genus Oscillateria Genus (Phaeodactylum), genus Platimonas (Playtomonas), genus Pleurochrysis, genus Pohyra, genus Pseudoanabaena, genus Pseudoanabaena, genus Pyramimonas, genus Pyramimonas Includes algae selected from the genus Synechopathy, the genus Tetraselmis, the genus Thalassiosira, and the genus Trichodesmium.

A portion of a plant, or "plant tissue," can be treated according to the method of the invention to produce an improved plant. Plant tissue also includes plant cells. The term "plant cell", as used herein, is an intact whole plant, or on medium or agar under in vitro tissue culture, in a suspension in growth medium or buffer, or Refers to individual units of living plants, either highly organized units, such as plant tissues, plant organs, or isolated forms grown as part of whole plants.

"Protoplasts" result in intact biochemically competent units of living plants that can reshape, proliferate and regenerate their cell walls to grow into whole plants under appropriate growth conditions. Refers to plant cells whose protective cell walls have been completely or partially removed, for example by mechanical or enzymatic means.

The term "transformation" broadly refers to a method in which a plant host is genetically modified by the introduction of DNA using Agrobacterium or one of a variety of chemical or physical methods. As used herein, the term "plant host" refers to a plant, including any cell, tissue, organ, or offspring of the plant. Many suitable plant tissues or plant cells can be transformed, including, but not limited to, protoplasts, somatic embryos, pollen, leaves, seedlings, stems, calluses, roots, microtubes, and seedlings. Plant tissue is also produced either sexually or asexually, such as such plants, seeds, offspring, any clone of propagules, and cuttings or seeds. Also refers to the descendants of.

The term "transformed", as used herein, refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of a recipient cell, tissue, organ, or organism so that the introduced DNA molecule is transmitted to subsequent offspring. In these embodiments, "transformed" or "transgenic" cells or plants are also produced and introduced from breeding schemes that use such transformed plants as parents, as well as the progeny of the cells or plants. Progeny that exhibit phenotypic changes caused by the presence of the DNA molecule can also be included. Preferably, the transgenic plant is fertile and is capable of transmitting the introduced DNA to offspring through sexual reproduction.

The term "offspring", such as the offspring of a transgenic plant, is born from, produced from, or derived from a plant or transgenic plant. The introduced DNA molecule may also be transiently introduced into the recipient cell, so that the introduced DNA molecule will not be inherited by subsequent offspring and is therefore not considered "transgenic". Thus, as used herein, a "non-transgenic" plant or plant cell is a plant that does not contain foreign DNA that is stably integrated into its genome.

The term "plant promoter", as used herein, is a promoter capable of initiating transcription in a plant cell, whether or not its origin is a plant cell. Exemplary suitable plant promoters include, but are not limited to, those obtained from bacteria such as Agrobacterium or Rhizobium, which contain genes expressed in plants, plant viruses, and plant cells. Can be mentioned.

As used herein, "fungal cell" refers to any type of eukaryotic cell within the fungal kingdom. The phylums within the fungal world include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Glomeromycota, and Glomeromycota. Microsporidia), and Neocallimastigomycota. Fungal cells can include yeast, mold, and filamentous fungi. In some embodiments, the fungal cell is a yeast cell.

As used herein, the term "yeast cell" refers to any fungal cell within the Ascomycota and Basidiomycota. Yeast cells can include budding yeast cells, fission yeast cells, and mold cells. Many types of yeast used in laboratory and industrial environments, but not limited to these organisms, are part of the Ascomycota. In some embodiments, the yeast cell is S. S. cerevisiae, Kluyveromyces marxianus, or Isachenkia orientalis cells. Other yeast cells include, without limitation, Candida spp. (Eg, Candida albicans), Yarrowia spp. (Eg, Yarrowia lipolic). , Pichia spp. (For example, Pichia pastoris), Kluyveromyces spp. (For example, Kluyveromyces lactis and Kluyveromyces lactis). Kluyveromyces marxianus), Candida albicans (Neurospora spp.) (Eg, Neurospora crassa), Fusarium spp. (For example, Fusarium spp.) (For example, Isachenkia orientalis, also known as Pichia kudriavzevii and Candida albicanthermofilm). In some embodiments, the fungal cell is a filamentous fungal cell. As used herein, the term "filamentous fungal cell" refers to any type of fungal cell that grows filamentous, ie, as a hypha or mycelium. Examples of filamentous fungal cells include, without limitation, Aspergillus spp. (For example, Aspergillus niger), Trichoderma spp. (Eg, Trichoderma spp.) (Eg, Trichoderma). ), Rhizopus spp. (For example, Rhizopus oryzae), and Mortierella spp. (For example, Mortierella isabella).

In some embodiments, the fungal cell is an industrial strain. As used herein, "industrial strain" refers to any strain of fungal cells used or isolated from an industrial process, eg, the production of a product on a commercial or industrial scale. .. An industrial strain may refer to a fungal species typically used in an industrial process, or it may refer to an isolate of a fungal species that can also be used for non-industrial purposes (eg, experimental studies). .. Examples of industrial processes include fermentation (eg, in the production of food or beverage products), distillation, biofuel production, compound production, and polypeptide production. Examples of industrial strains include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As used herein, a "multiply" cell can refer to any cell in which the genome resides in more than one copy. A polyploid cell may refer to a type of cell that is naturally found in the polyploid state, or as it exists in the polyploid state (eg, specific regulation of meiosis, cytokinesis, or DNA replication, It may refer to cells induced (by alteration, inactivation, activation, or modification). A polyploid cell may refer to a cell whose entire genome is haploid, or it may refer to a cell which is haploid at a genomic locus of particular interest. Although not bound by theory, it is described herein because the abundance of guide RNA can be the rate-determining component in more cases in genomic engineering of haploid cells compared to haploid cells. It is believed that the method using the CRISPR / Cas9 system to be used can take advantage of using a particular fungal cell type.

In some embodiments, the fungal cell is a diploid cell. As used herein, a "diploid" cell can refer to any cell in which the genome resides in two copies. A diploid cell may refer to a type of cell that is naturally found in a diploid state, or as it exists in a diploid state (eg, meiosis, cytokinesis, or identification of DNA replication. It may refer to cells induced (by regulation, alteration, inactivation, activation, or modification of). For example, S. S. cerevisiae strain S228C can be maintained in a haploid or diploid state. A diploid cell may refer to a cell whose entire genome is diploid, or it may refer to a cell which is diploid at a genomic locus of particular interest. In some embodiments, the fungal cell is a haploid cell. As used herein, a "haploid" cell can refer to any cell in which the genome resides in one copy. A haploid cell may refer to a type of cell that is naturally found in the haploid state, or as it exists in the haploid state (eg, meiosis, cytokinesis, or identification of DNA replication. It may refer to cells induced (by regulation, alteration, inactivation, activation, or modification of). For example, S. S. cerevisiae strain S228C can be maintained in a haploid or diploid state. A haploid cell may refer to a cell whose entire genome is haploid, or it may refer to a cell which is haploid at a genomic locus of particular interest.

As used herein, a "yeast expression vector" is a nucleic acid containing one or more sequences encoding RNA and / or a polypeptide, and controls the expression of one or more nucleic acids thereof. Refers to a nucleic acid that may further contain any desired element to be added, as well as any element that allows the replication and maintenance of the expression vector inside the yeast cell. Many suitable yeast expression vectors and their characteristics are known in the art; for example, various vectors and techniques are available from Yeast Protocols, 2nd edition, Xiao, W. et al. , Ed. (Humana Press, New York, 2007) and Buckholz, R. et al. G. and Greeson, M.D. A. (1991) Biotechnology (NY) 9 (11): 1067-72. Yeast vectors are, without limitation, promoters such as centromere (CEN) sequences, self-replicating sequences (ARS), RNA polymerase III promoters operably linked to the sequence or gene of interest, terminators such as RNA polymerase III terminators, replication. It may contain an origin and a marker gene (eg, a terminator, an antibiotic, or other selectable marker). Examples of expression vectors used in yeast include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integration plasmids, yeast replication plasmids, shuttle vectors, and episomal plasmids.

Stable integration of CRISPR / Cas9 system components in plant and plant cell genomes In a detailed embodiment, it is assumed that a polynucleotide encoding a CRISPR / Cas9 system component is introduced for stable integration into the plant cell genome. To. In these embodiments, the design of the transformation vector or expression system can be tailored depending on when, where, and under what conditions the chi / sgRNA and / or Cas9 gene is expressed.

In a detailed embodiment, it is assumed that the components of the CRISPR / Cas9 system are stably introduced into the genomic DNA of plant cells. In addition to or instead, it is envisioned that components of the CRISPR / Cas9 system will be introduced for stable integration into the DNA of plant organelles such as, but not limited to, plastids, mitochondria or chloroplasts.

An expression system for stable integration into the plant cell genome can contain one or more of the following elements: promoter elements that can be used to express RNA and / or Cas9 enzymes in plant cells; enhance expression. 5'untranslated region; intron element that further enhances expression in certain cells, such as monocotic plant cells; provides a convenient restriction site for inserting the chi / sgRNA and / or Cas9 gene sequence and other desired elements. Multiple cloning sites; and 3'untranslated regions that result in efficient termination of the expressed transcript.

The elements of the expression system may be on one or more expression constructs that are either circular, such as a plasmid or transformation vector, or acyclic, such as linear double-stranded DNA. In a detailed embodiment, the CRISPR-Cas9 expression system is at least:
(A) A nucleotide sequence encoding a guide or chi / sgRNA that hybridizes with a plant target sequence, wherein the guide or chi / sgRNA comprises a guide sequence and a direct repeat sequence, and (b) Cas9 protein. Nucleotide sequence encoding,
Including
Here, components (a) or (b) are located on the same or different constructs, and thereby different nucleotide sequences can be under the control of the same or different regulatory elements that can operate in plant cells.

One or more DNA constructs containing components of the CRISPR / Cas9 system and, where applicable, template sequences can be introduced into the genome of a plant, plant site, or plant cell by a variety of prior art techniques. This process generally involves selecting a suitable host cell or tissue, introducing one or more constructs into the host cell or host tissue, and then regenerating the plant cell or plant. In a detailed embodiment, the DNA construct may be introduced into the plant cell using techniques such as, but not limited to, electroporation, microinjection, and aerosol beam injection of the plant cell protoplast, or the DNA construct is a DNA particle. It can also be introduced directly into plant tissue using a micro-gun method such as bombardment (see also Fu et al., Transgenic Res. 2000 Feb; 9 (1): 11-9). The basis of particle bombardment is that particles coated with one or more genes of interest are accelerated toward the cell to allow the particles to invade the protoplasm and typically obtain stable integration into the genome. (See, eg, Klein et al, Nature (1987), Klein et ah, Bio / Technology (1992), Casa et ah, Proc. Natl. Acad. Sci. USA (1993)).

In a detailed embodiment, DNA constructs containing components of the CRISPR / Cas9 system can be introduced into plants by Agrobacterium-mediated transformation. The DNA construct can be combined with a suitable T-DNA flanking region and introduced into a conventional Agrobacterium tumefaciens host vector. Foreign DNA is incorporated into the plant genome either by infecting the plant or by incubating the plant protoplast with one or more Ti (tumor-inducing) plasmid-containing Agrobacterium bacteria. (See, eg, Flatley et al., (1985), Rogers et al., (1987) and US Pat. No. 5,563,055).

Plant Promoters To ensure proper expression in plant cells, the components of the CRISPR / Cas9 system described herein are typically placed under the control of a plant promoter, i.e., a promoter operable in plant cells. Be taken. The use of different types of promoters is envisioned.

A constitutive plant promoter causes the open reading frame (ORF) it controls to be expressed in all or almost all plant tissues at all or almost every developmental stage of a plant (referred to as "constitutive expression"). Is a possible promoter. One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. "Regulatory promoter" refers to a promoter that induces gene expression in a non-constitutive but temporally and / or spatially regulated manner, and includes tissue-specific, tissue-preferred and inducible promoters. Different promoters can induce gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental conditions. In a detailed embodiment, one or more of the CRISPR / Cas9 components are expressed under the control of a constitutive promoter such as the cauliflower mosaic virus 35S promoter and are present within a particular plant tissue by utilizing a tissue-preferred promoter. Enhanced expression in the cell type of the species, eg, leaf or root vascular cells or seed specific cells, can be targeted. For examples of detailed promoters used in the CRISPR / Cas9 system, see Kawamata et al. , (1997) Plant Cell Physiol 38: 792-803; Yamamoto et al. , (1997) Plant J 12: 255-65; Hire et al, (1992) Plant Mol Biol 20: 207-18, Kuster et al, (1995) Plant Mol Biol 29: 759-72, and Capana et al. , (1994) Plant Mol Biol 25: 681-91. Examples of promoters that are inducible and capable of spatiotemporally controlling gene editing or gene expression can use certain forms of energy. The form of energy may include, but is not limited to, sound energy, electromagnetic radiation, chemical energy and / or thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet on or Tet off), small molecule two-hybrid transcriptional activation systems (FKBP, ABA, etc.), or photoinducible systems (phytochrome, LOV domain, or cryptochrome). For example, a photo-induced transcription effector (LITE) that induces a sequence-specific change in transcription activity. Components of the photo-inducible system can include the CRISPR / Cas9 enzyme, photoresponsive cytochrome heterodimers (eg from Arabidopsis thaliana), and transcriptional activation / repressor domains. Further examples of inducible DNA-binding proteins and their uses are incorporated herein by reference in US Provisional Patent Application No. 61/736465 and US Provisional Patent Application No. 61 / 721,283 (as a whole by reference herein). Will be provided).

In a detailed embodiment, transient or inducible expression can be achieved, for example, using a chemical-regulated promoter, that is, the addition of an exogenous chemical induces gene expression. Regulation of gene expression can also be achieved by chemical inhibitory promoters, where the addition of chemicals suppresses gene expression. Chemical-inducible promoters include, but are not limited to, the corn ln2-2 promoter activated by a benzenesulfonamide herbicide detoxifier (De Velder et al., (1997) Plant Cell Physiol 38: 568-77), The corn GST promoter (GST-II-27, WO 93/01294) activated by hydrophobic electrophobic compounds used as pre-germ herbicides, and the tobacco PR-1a promoter (Ono) activated by salicylic acid. et al., (2004) Bioscii Biotechnol Biochem 68: 803-7). Antibiotic-regulated promoters, such as the tetracycline-inducible and tetracycline-suppressing promoters, are also (Gatz et al., (1991) Mol Gen Genet 227: 229-37; US Pat. No. 5,814,618 and the same. 5,789,156), which can be used herein.

Translocation to and / or expression in a particular plant organelle The expression system may include elements for translocation and / or expression in a particular plant organelle.

Chloroplast targeting In a detailed embodiment, it is envisioned that the CRISPR / Cas9 system will be used to specifically modify the chloroplast gene or ensure expression in the chloroplast. For this purpose, a chloroplast transformation method or compartmentalization of CRISPR / Cas9 system components into chloroplasts is used. For example, introducing genetic modification into the plastid genome can alleviate biosafety problems such as gene flow through pollen.

Chloroplast transformation methods are known in the art and include particle bombardment, PEG treatment, and microinjection. In addition, methods involving transposition of transformation cassettes from the nuclear genome to plastids can be used, as described in WO 201406186.

Alternatively, it is envisioned that one or more of the CRISPR / Cas9 system components will be targeted to plant chloroplasts. This is accomplished by incorporating into the expression construct a sequence encoding a chloroplast transport peptide (CTP) or plastid transport peptide operably linked to the 5'region of the Cas9 protein-encoding sequence. CTP is removed at the processing step during translocation to chloroplasts. Chloroplast targeting of expressed proteins is well known to those of skill in the art (see, eg, Protein Transport into Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61: 157-180). In such embodiments, it is also desirable to target the chi / sgRNA to plant chloroplasts. Methods and constructs that can be used to translocate chi / sgRNA to chloroplasts using chloroplast localized sequences are described, for example, in US Patent Application Publication No. 20040142476 (see herein by reference). Incorporated). By incorporating such various constructs into the expression system of the present invention, Cas9-chi / sgRNA can be efficiently translocated.

Introduction of polynucleotides encoding the CRISPR-Cas9 system in algae cells Transgenic algae (or other plants, such as Rapeseed) are used in the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol) or other products. Can be particularly useful. They can be engineered to highly express or overexpress the oils or alcohols used in the oil or biofuel industry.

US Pat. No. 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii) species using Cas9. Similarly, the CRISPR / Cas9 series described herein can be applied to Chlamydomonas species and other algae. In a detailed embodiment, Cas9 and chi / sgRNA are introduced into algae expressed using a vector that expresses Cas9 under the control of a constitutive promoter such as Hsp70A-Rbc S2 or β2-tubulin. The chi / sgRNA is optionally delivered using a vector containing the T7 promoter. Alternatively, Cas9 mRNA and in vitro transcription chi / sgRNA may be delivered to algae cells. Electroporation protocols are available to those of skill in the art, such as the standard recommended protocol from the GeneArt Chlamydomonas Engineering Kit.

In a detailed embodiment, the endonuclease used herein is a split Cas9 enzyme. The split Cas9 enzyme is preferentially used in algae for targeted genomic modification, as described in WO 2015086795. The use of the Cas9 split system is particularly suitable for inducible genomic targeting methods, avoiding the potential toxic effects of Cas9 overexpression in algal cells. In a detailed embodiment, the Cas9 split domains (RuvC and HNH domains) are introduced into cells simultaneously or sequentially so that the one or more split Cas9 domains process the target nucleic acid sequence in the algae cells. Can be done. Due to the smaller size of split Cas9 compared to wild-type Cas9, other methods of delivering the CRISPR system to cells are possible, such as the use of cell-permeable peptides as described herein. This method is particularly useful for the production of genetically modified algae.

Introducing a polynucleotide encoding a Cas9 component in yeast cells In a detailed embodiment, the present invention relates to the use of the CRISPR / Cas9 system for genome editing in yeast cells. Yeast cell transformation methods that can be used to introduce polynucleotides encoding CRISPR / Cas9 system components are well known in the art and are known in Kawai et al. , 2010, Bioeng Bugs. It has been reviewed by 2010 Nov-Dec; 1 (6): 395-403). Non-limiting examples include transformation of yeast cells by lithium acetate treatment, which may further include carrier DNA and PEG treatment, bombardment or electroporation.

Transient expression of Cas9 CRISP system components in plants and plant cells In a detailed embodiment, it is assumed that chi / sgRNA and / or Cas9 gene is transiently expressed in plant cells. In these embodiments, the CRISPR / Cas9 system ensures modification of the target gene only when both the chi / sgRNA and Cas9 protein are present in the cell, thus allowing further control of genomic modification. Since the expression of Cas9 enzyme is transient, plants regenerated from such plant cells typically do not contain foreign DNA. In a detailed embodiment, the Cas9 enzyme is stably expressed by plant cells, and the guide sequence is transiently expressed.

In a detailed embodiment, the CRISPR / Cas9 system components can be introduced into plant cells using a plant viral vector (Scholthof et al. 1996, Annu Rev Phytopasol. 1996; 34: 299-323). In a more detailed embodiment, the viral vector is a vector derived from a DNA virus. For example, Gemini virus (eg, cabbage roll virus, bean yellow virus, wheat dwarf virus, tomato roll virus, corn streak virus, tobacco roll leaf virus, or tomato golden mosaic virus) or nanovirus (eg, soramame yellowing). Eso virus). In another detailed embodiment, the viral vector is an RNA virus-derived vector. For example, Tobra virus (eg, tobacco stem virus, tobacco mosaic virus), Portex virus (eg, potato X virus), or Holdy virus (eg, wheat mottled mosaic virus). The replication genome of plant viruses is a non-integrated vector.

In a detailed embodiment, the vector used for transient expression of the CRISPR / Cas9 construct is, for example, the pEAQ vector, which is tuned for Agrobacterium-mediated transient expression in protoplasts. (Sainsbury F. et al., Plant Biotechnol J. 2009 Sep; 7 (7): 682-93). Accurate targeting of genomic positions has been demonstrated using a modified cabbage roll virus (CaLCV) vector that expresses gRNA in stable transgenic plants that express the CRISPR enzyme (Scientific Reports 5, Article number: 14926 (2015)). , Doi: 10.1038 / rep14926).

In a detailed embodiment, a double-stranded DNA fragment encoding the chi / sgRNA and / or Cas9 gene can be transiently introduced into a plant cell. In such embodiments, the introduced double-stranded DNA fragment is provided in an amount sufficient to modify the cell, but not remaining after the intended time or after one or more cell divisions. Methods of deriving DNA transfer in plants are known to those of skill in the art (see, eg, Davey et al. Plant Mol Biol. 1989 Sep; 13 (3): 273-85).

In other embodiments, the RNA polynucleotide encoding the Cas9 protein is sufficient to modify the cell (in the presence of at least one chi / sgRNA), but after the intended time or more than once. Is introduced into plant cells in an amount that does not remain after cell division, which is then translated and processed by the protein-producing host cell. Methods of introducing mRNA into plant protoplasts for transient expression are known to those of skill in the art (see, eg, Gallie, Plant Cell Reports (1993), 13; 119-122).
A combination of the different methods described above is also envisioned.

Delivery of CRISPR / Cas9 Components to Plant Cells In a detailed embodiment, it is beneficial to deliver one or more components of the CRISPR / Cas9 system directly to plant cells. This is particularly beneficial for the production of non-transgenic plants (see below). In a detailed embodiment, one or more of the Cas9 components are prepared outside the plant or plant cell and delivered to the cell. For example, in a detailed embodiment, the Cas9 protein is prepared in vitro and then introduced into plant cells. Cas9 protein can be prepared by various methods known to those skilled in the art, including recombinant production. After expression, the Cas9 protein is isolated, refolded and purified as needed, and optionally processed to remove any purified tags such as His tags. Once a crude, partially purified, or more fully purified Cas9 protein is obtained, the protein can be introduced into plant cells.

In a detailed embodiment, the Cas9 protein is mixed with a chi / sgRNA that targets the gene of interest to form a pre-assembled ribonucleoprotein.

Individual components or pre-assembled ribonuclear proteins can be transported by electroporation, by bombardment of Cas9-related gene-coated particles, by chemical transfection, or across cell membranes. It can be introduced into plant cells by some means. For example, transfection of plant protoplasts with pre-assembled CRISPR ribonuclear proteins has been demonstrated to ensure targeted alterations of the plant genome (Woo et al. Nature Biotechnology, 2015; DOI: 10.1038). (As described by / nbt.3389).

In a detailed embodiment, the CRISPR / Cas9 system components are introduced into plant cells using nanoparticles. The constituents, whether as proteins or nucleic acids, or combinations thereof, can be uploaded on nanoparticles or packaged in them for plant applications (eg, WO 2008042156 and US patent applications). Publication No. 201301858223, etc.). In particular, embodiments of the present invention encode one or more DNA molecules, chi / sgRNA and / or isolated chi / sgRNA, encoding the Cas9 protein, as described in WO 2015089419. Contains nanoparticles in which the DNA molecule has been uploaded or packaged with it.

A further means of introducing one or more components of the CRISPR / Cas9 system into plant cells is by using a cell-permeable peptide (CPP). Thus, in particular, embodiments of the invention include compositions comprising cell-permeable peptides linked to Cas9 proteins. In a detailed embodiment of the invention, Cas9 proteins and / or chi / sgRNAs are coupled to one or more CPPs that effectively transport them into plant protoplasts (for Cas9 in human cells, Ramakrishana (for Cas9 in human cells). (As described by 2014 Genome Res. 2014 Jun; 24 (6): 1020-7). In other embodiments, the Cas9 gene and / or chi / sgRNA is added to one or more CPPs for plant protoplast delivery. Encoded by one or more coupled circular or acyclic DNA molecules. The plant protoplasts are then regenerated into plant cells and even plants. CPPs are generally receptor-independent of biomolecules. CPPs are described as short chain peptides of less than 35 amino acids derived from either proteins or chimeric sequences capable of transporting across cell membranes. CPPs are cationic peptides, peptides with hydrophobic sequences, protoplastic peptides. , Peptides with protopyl-rich antimicrobial sequences, and may be chimeric or bipartite peptides (Pooga and Langel 2005). CPPs are capable of penetrating biomembranes and thus of various biomolecules. It is possible to cause migration across the cell membrane into the cytoplasm and to improve their intracellular transport, thus facilitating the interaction of biomolecules with targets. Examples of CPPs include, among others, HIV type 1. Tat, penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence; polyarginine peptide Args sequence, guanine-rich molecule transporter, sweet Arrow peptides and the like can be mentioned.

Generating non-transgenic plants using the CRISPR / Cas9 system In a detailed embodiment, the methods described herein include any exogenous genes, including those encoding CRISPR components, in the genome of the plant. It is used to modify endogenous genes or modify their expression, thus avoiding the presence of foreign DNA in the plant genome, without permanent introduction into. This can be beneficial as the regulatory requirements for non-transgenic plants are less stringent.

In a detailed embodiment, this is ensured by transient expression of the CRISPR / Cas9 component. In a detailed embodiment, one or more of the CRISPR components produce enough Cas9 protein and chi / sgRNA to consistently and consistently reliably modify the gene of interest by the methods described herein. Expressed on one or more viral vectors.

In a detailed embodiment, transient expression of the CRISPR / Cas9 construct occurs reliably in plant protoplasts and therefore does not integrate into the genome. A limited expression window may be sufficient to allow the CRISPR / Cas9 system to reliably modify the target gene as described herein.

In a detailed embodiment, the various components of the CRISPR / Cas9 system are separated or mixed with the help of particulate delivery molecules such as nanoparticles or CPP molecules as described above herein. Then, it is introduced into plant cells, protoplasts or plant tissues.

When the CRISPR / Cas9 component is expressed, direct activity of the Cas9 nuclease and optionally the introduction of template DNA, or modification of the targeted gene using the CRISPR / Cas9 system as described herein. Can induce targeted modification of the genome. The various strategies described above herein allow Cas9-mediated target genome editing without the need to introduce CRISPR / Cas9 components into the plant genome. Components that are transiently introduced into plant cells are typically removed during mating.

Detection of Modifications in the Plant Genome-Selectable Markers In a detailed embodiment, if the method involves an endogenous target gene modification of the plant genome, after infecting or transfecting a plant, plant site or plant cell with the CRISPR / Cas9 system. , Any suitable method can be used to determine whether gene targeting or target mutagenesis occurs at the target site. If the method involves the introduction of a transgene, identify the transformed plant cell, callus, tissue or plant by selecting or screening the engineered plant material for the presence of the transgene or the trait encoded by the transgene. And can be isolated. Physical and biochemical methods can be used to identify plants or plant cell transformants containing the inserted gene constructs or endogenous DNA modifications. These methods include, but are not limited to: 1) Southern analysis or PCR amplification to detect and determine the structure of recombinant DNA inserts or modified endogenous genes; 2) Detect and examine RNA transcripts of gene constructs. Northern blot, S1 RNase protection, primer extension or reverse transcription enzyme PCR amplification; 3) Enzyme assay to detect enzyme or ribozyme activity (such gene product is encoded by a gene construct or expression is affected by genetic modification Case); 4) Protein gel electrophoresis, Western blot, immunoprecipitation, or enzyme-bound immunoassay (when the gene construct or endogenous gene product is a protein). Further techniques such as in situ hybridization, enzymatic staining, and immunostaining can also be used to detect the presence or expression of recombinant constructs, or to detect alterations of endogenous genes in certain plant organs and tissues. Methods of performing all these assays are well known to those of skill in the art.

In addition (or instead), expression systems encoding CRISPR / Cas9 components typically include cells containing the CRISPR / Cas9 system and / or cells modified thereby at an early stage and on a large scale. Is designed to include one or more selectable or detectable markers that provide a means of isolation or efficient selection.

For Agrobacterium-mediated transformation, the marker cassette may be adjacent to or between the flanking T-DNA boundaries and be contained within the binary vector. In another embodiment, the marker cassette may be outside the T-DNA. The selectable marker cassette may also be in or adjacent to the same T-DNA boundary as the expression cassette, or somewhere else in the second T-DNA on a binary vector (eg, 2T-DNA system). It may be at the place.

For particle bombardment, or for protoplast transformation, the expression system can contain one or more isolated linear fragments, or contains a bacterial replication element, a bacterial selectable marker or other detectable element. It may be part of a large structure to be obtained. One or more expression cassettes containing the guide and / or the polynucleotide encoding Cas9 may be physically linked to the marker cassette or may be mixed with a second nucleic acid molecule encoding the marker cassette. .. The marker cassette contains elements essential for the expression of detectable or selectable markers that allow efficient selection of transformed cells.

The procedure for selecting cells based on selectable markers will depend on the nature of the marker gene. In a detailed embodiment, a selectable marker, i.e., a marker that allows cells to be selected directly based on the expression of the marker is used. Selectable markers can result in positive or negative selection and are conditioned or unconditional with respect to the presence of external substrates (Miki et al. 2004,107 (3): 193-232). Most commonly, an antibiotic or herbicide resistance gene is used as a marker, thereby engineering on a medium containing an inhibitory amount of the antibiotic or herbicide subject to resistance conferred by the marker gene. Selection is made by growing plant material. Examples of such genes include genes that confer antibiotic resistance, such as hygromycin (hpt) and kanamycin (nptII), and herbicide resistance, such as phosphinotricine (bar) and chlorosulfuron (als). It is a gene to be given.

Transformed plants and plant cells may also be identified by screening for the activity of enzymes capable of processing visible markers, typically colored substrates (eg, β-glucuronidase, luciferase, B or C1 gene). Good. Such selection and screening methods are well known to those of skill in the art.

Plant Cultures and Regeneration In detailed embodiments, plant cells that have a modified genome and are produced or obtained by any of the methods described herein are transformed or modified by culturing. All plants with the same genotype and thus with the desired phenotype can be regenerated. Conventional regeneration techniques are well known to those of skill in the art. A detailed example of such a regeneration technique relies on the manipulation of certain plant hormones in tissue culture growth media, and typically biocides and / or herbicides introduced with the desired nucleotide sequence. It relies on markers. In a more detailed embodiment, plant regeneration is obtained from cultured protoplasts, plant calluses, explants, organs, pollen, embryos or parts thereof (eg, Evans et al. (1983), Handbook of Plant). Cell Culture, Klee et al (1987) Ann. Rev. of Plant Phys).

In a detailed embodiment, the transformed or modified plant as described herein provides or does not provide seeds of the homozygous improved plant of the invention (homozygous with respect to DNA modification) by self-pollination. Heterozygous plant seeds can be provided by crossing with a transgenic plant or another modified plant. When recombinant DNA is introduced into a plant cell, the plant obtained by such crossing is a heterozygous plant with respect to the recombinant DNA molecule. Both homozygous and heterozygous plants, including genetically modified (possibly recombinant DNA), obtained by crossing from improved plants are referred to herein as "offspring." Offspring plants are plants derived from the original transgenic plant and containing genomically modified or recombinant DNA molecules introduced by the methods provided herein. Alternatively, the genetically modified plant can be obtained by one of the above-mentioned methods using Cas9 in which foreign DNA is not incorporated into the genome. Offspring of such plants obtained by further breeding may also contain the genetic modification. Breeding is carried out by any breeding method commonly used for various crops (eg, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U.of CA, Davis, CA, 50-98 (eg). 1960).

Generation of Plants with Enhanced Agricultural Traits The Cas9-based CRISPR system provided herein can be used to introduce targeted double- or single-strand breaks and / or gene activators and / or repressors. The system can be introduced and, without limitation, used for gene targeting, gene substitution, target mutagenesis, target deletion or insertion, target inversion and / or target translocation. Co-expression of multi-targeting RNA performed to achieve multiple modifications in a single cell ensures that the multiplexed genome modification can be performed. This technique provides a high level of plants with improved characteristics, including increased nutritional value, disease resistance and resistance to biological and abiotic stress, and increased production of commercially useful plant products or heterologous compounds. It can be used for precision engineering.

In a detailed embodiment, a target double-strand break (DSB) is introduced into the endogenous DNA sequence using the CRISPR / Cas9 system as described herein. The DSB activates the cellular DNA repair pathway, which can be used to achieve the desired DNA sequence modification in the vicinity of the cleavage site. This is useful if inactivation of the endogenous gene can confer or contribute to the desired trait. In a detailed embodiment, homologous recombination with the template sequence is promoted at the site of DSB in order to introduce the gene of interest.

In a detailed embodiment, the CRISPR / Cas9 system can be used as a common nucleic acid binding protein fused or operably linked to a functional domain for activating and / or suppressing an endogenous plant gene. Exemplary functional domains include, but are not limited to, translation initiation factors, translation activators, translational suppressors, nucleases, and more specifically ribonucleases, spliceosomes, beads, photoinducible / regulatory domains or chemical-inducible / Controllable domains can be mentioned. Typically, in these embodiments, the Cas9 protein contains at least one mutation, so that it has less than 5% activity of the Cas9 protein without at least one mutation; the chi / sgRNA is the target. The sequence contains a hybridizable guide sequence.

The methods described herein generally result in the production of "improved plants" in that they have one or more desirable traits compared to wild-type plants. In a detailed embodiment, the resulting plant, plant cell or plant site is a transgenic plant and comprises an exogenous DNA sequence incorporated into the genome of all or part of the plant cell. In a detailed embodiment, a non-transgenic genetically modified plant, plant site or cell is obtained in that no exogenous DNA sequence is incorporated into the genome of any plant cell of the plant. In such embodiments, the modified plant is non-transgenic. If only modification of the endogenous gene occurs reliably and the exogenous gene is not introduced or maintained in the plant genome, the resulting genetically modified crop does not contain the exogenous gene and can therefore be considered essentially non-transgenic. Various applications of the CRISPR / Cas9 system to plant genome editing will be described in more detail below.

a) Conferring a desired agricultural trait by introducing one or more exogenous genes The present invention provides a method for genome editing or modifying a sequence associated with or located at a target locus of interest, which method Cas9. The step of introducing the effector protein complex into a plant cell, whereby the Cas9 effector protein complex functions effectively for the integration of a DNA insert (eg, encoding an exogenous gene of interest) into the genome of the plant cell. including. In a preferred embodiment, the integration of the DNA insert is facilitated by HR with an extrinsically introduced DNA template or repair template. Typically, the exogenously introduced DNA template or repair template is delivered with a Cas9 effector protein complex or a polynucleotide vector expressing a component or component of the complex.

The CRISPR / Cas9 system provided herein allows for target gene delivery. It is becoming increasingly clear that the expression efficiency of the gene of interest is largely determined by the position of integration into the genome. The method allows the exogenous gene to be targeted and integrated at a desired location in the genome. The location can be selected based on information on events that have occurred so far, or can be selected by the methods disclosed elsewhere herein.

In a detailed embodiment, the method provided herein is a step of introducing (a) a CRISPR / Cas9 complex containing a chi / sgRNA (including a direct repeat and a guide sequence) into a cell, the plant. Steps in which the guide sequence hybridizes to the target sequence endogenous to the cell; (b) When the guide sequence hybridizes to the target sequence, it forms a complex with chi / sgRNA and is located at or near the target sequence of the guide sequence. Steps of introducing a Cas9 effector molecule that induces double-strand breaks into plant cells; and (c) a nucleotide sequence that encodes the gene of interest and encodes an HDR repair template that is introduced at the DS break position as a result of HDR. Includes steps to introduce into cells. In a detailed embodiment, the step of introduction may include delivering one or more polynucleotides encoding a Cas9 effector protein and a chi / sgRNA to a repair template to a plant cell. In a detailed embodiment, the polynucleotide is delivered to the cell by a DNA virus (eg, Geminivirus) or an RNA virus (eg, Tobravirus). In a detailed embodiment, the step of introduction comprises delivering to plant cells a T-DNA containing one or more polynucleotide sequences encoding a Cas9 effector protein and a chi / sgRNA and a repair template. Delivery is by the genus Agrobacterium. The nucleic acid sequence encoding the Cas9 effector protein may be operably linked to a promoter, such as a constitutive promoter (eg, cauliflower mosaic virus 35S promoter), or a cell-specific or inducible promoter. In a detailed embodiment, the polynucleotide is introduced by microprojectile bombardment. In a detailed embodiment, the method further comprises screening plant cells after the step of introduction to determine a repair template, i.e., whether the gene of interest has been introduced. In a detailed embodiment, the method comprises the step of regenerating a plant from a plant cell. In a further embodiment, the method comprises cross breeding the plant to obtain a genetically desirable plant line. Examples of exogenous genes encoding the trait of interest are given below.

b) Addition of a desired agricultural trait by editing an endogenous gene The present invention provides a method for genome editing or modification of a sequence associated with or located at a target locus of interest, which method is a Cas9 effector protein complex. Is introduced into plant cells, thereby comprising the step of modifying the expression of the plant's endogenous gene by the Cas9 complex. This can be achieved in a variety of ways, and in detailed embodiments it is desirable to eliminate the expression of the endogenous gene, by targeting and cleaving the endogenous gene with the CRISPR / Cas9 complex. Is modified. In these embodiments, the methods provided herein are: (a) Introducing a CRISPR / Cas9 complex containing a chi / sgRNA (including a direct repeat and a guide sequence) into a plant cell [the guide sequence is Hybridizes to a target sequence within a gene of interest in the genome of a plant cell]; and (b) contains a guide sequence that hybridizes to the target sequence upon binding to chi / sgRNA, and the guide sequence is the target sequence or a sequence thereof. Including the step of introducing a Cas9 effector protein into a cell that ensures double-strand breaks in the vicinity; in a detailed embodiment, the step of introducing the Cas9 effector protein and one or more polynucleotides encoding a chi / sgRNA. It may include delivery to plant cells.

In a detailed embodiment, the polynucleotide is delivered to the cell by a DNA virus (eg, Geminivirus) or an RNA virus (eg, Tobravirus). In a detailed embodiment, the step of introduction comprises delivering T-DNA containing one or more polynucleotide sequences encoding Cas9 effector proteins and chi / sgRNA to plant cells, where delivery is Agrobacterium. According to the genus Agrobacterium. The polynucleotide sequence encoding a component of the CRISPR / Cas9 system may be operably linked to a promoter, such as a constitutive promoter (eg, cauliflower mosaic virus 35S promoter), or a cell-specific or inducible promoter. In a detailed embodiment, the polynucleotide is introduced by microprojectile bombardment. In a detailed embodiment, the method further comprises screening plant cells after the step of introduction to determine if the expression of the gene of interest has been altered. In a detailed embodiment, the method comprises the step of regenerating a plant from a plant cell. In a further embodiment, the method comprises cross breeding the plant to obtain a genetically desirable plant line.

In a detailed embodiment of the method described above, disease resistant crops are obtained by targeted mutation of a gene encoding a morbidity gene or a negative regulator of a plant defense gene (eg, the Mlo gene). In a detailed embodiment, herbicide-tolerant crops are produced by targeted replication of specific nucleotides in plant genes, such as those encoding acetolactate synthase (ALS) and protoporphyrinogen oxidase (PPO). In a detailed embodiment, drought-tolerant and salt-tolerant crops due to targeted mutations in genes encoding negative regulators of abiotic stress resistance, low amylose grains due to targeted mutations in the Waxy gene, major lipase genes in the aleurone layer Low acid defeating rice or other grains due to target mutation. In a detailed embodiment. Below is a more comprehensive list of endogenous genes encoding the trait of interest.

c) Conferring the desired agricultural trait by regulating the endogenous gene with the CRISPR / Cas9 system Also, herein, the Cas9 protein provided herein is used to regulate (ie, activate or activate) the endogenous gene expression. A method of suppressing) is also provided. Such a method utilizes one or more individual RNA sequences targeted to the plant genome by the Cas9 complex. More specifically, one or more individual RNA sequences bind to two or more adapter proteins (eg, aptamers), whereby each adapter protein associates with one or more functional domains, and here the adapter protein. At least one of the one or more functional domains associated with is methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, DNA integration activity, RNA cleavage activity, DNA cleavage activity. Alternatively, it has one or more activities, including nucleic acid binding activity; functional domains are used to regulate the expression of endogenous plant genes to obtain the desired trait. Typically, in these embodiments, the Cas9 effector protein has one or more mutations and thus has less than 5% nuclease activity of the Cas9 effector protein without at least one mutation.

In a detailed embodiment, the method provided herein is (a) the step of introducing a CRISPR / Cas9 complex containing a chi / sgRNA (including a direct repeat and a guide sequence) into a cell [the guide sequence is a plant]. Hybridizes to a target sequence endogenous to the cell]; (b) includes the step of introducing a Cas9 effector molecule that forms a complex with chi / sgRNA into the plant cell when the guide sequence hybridizes to the target sequence; and here. Either the chi / sgRNA has been modified to contain a separate RNA sequence (aptamer) that binds to the functional domain, and / or the Cas9 effector protein has been modified in that it is linked to the functional domain. Is. In a detailed embodiment, the step of introduction may include delivering one or more polynucleotides encoding (modified) Cas9 effector protein and (modified) chi / sgRNA to plant cells. Details of the components of the CRISPR / Cas9 system used in these methods are described elsewhere herein.

In a detailed embodiment, the polynucleotide is delivered to the cell by a DNA virus (eg, Geminivirus) or an RNA virus (eg, Tobravirus). In a detailed embodiment, the step of introduction comprises delivering T-DNA containing one or more polynucleotide sequences encoding Cas9 effector proteins and chi / sgRNA to plant cells, where delivery is Agrobacterium. According to the genus Agrobacterium. Nucleic acid sequences encoding one or more components of the CRISPR / Cas9 system may be operably linked to a promoter, such as a constitutive promoter (eg, cauliflower mosaic virus 35S promoter), or a cell-specific or inducible promoter. .. In a detailed embodiment, the polynucleotide is introduced by microprojectile bombardment. In a detailed embodiment, the method further comprises screening plant cells after the step of introduction to determine if the expression of the gene of interest has been altered. In a detailed embodiment, the method comprises the step of regenerating a plant from a plant cell. In a further embodiment, the method comprises cross breeding the plant to obtain a genetically desirable plant line. Below is a more comprehensive list of endogenous genes encoding the trait of interest.

Use of Cas9 to modify polyploid plants Many plants are polyploid, meaning that they have double copies of their genome (sometimes as many as six, as in wheat). is there. The method according to the invention utilizing the CRISPR / Cas9 effector protein is "multiplexed" so that it can affect all copies of a gene or target dozens of genes at once. .. For example, in a detailed embodiment, the methods of the invention are used to ensure that loss-of-function mutations occur simultaneously in various genes involved in suppressing defense against disease. In a detailed embodiment, the method of the present invention is used to simultaneously suppress the expression of TaMLO-Al, TaMLO-Bl and TaMLO-Dl nucleic acid sequences in wheat plant cells, and then the wheat plant is regenerated to make the wheat plant powdery mildew. It is ensured to be resistant to this disease (see also International Publication No. 2015109752).

Illustrative Genes Conferring Agricultural Traits As described above herein, in detailed embodiments, the present invention relates to the present specification for the insertion of a DNA of interest, including one or more plant-expressing genes. Includes the use of CRISPR / Cas9 systems as described in the document. In a more detailed embodiment, the invention includes methods and tools using the Cas9 system as described herein for partial or complete deletion of one or more plant expression genes. To do. In another further detailed embodiment, the invention modifies one or more plant-expressed genes by mutation, substitution, or insertion of one or more nucleotides using the Cas9 system as described herein. Includes methods and tools to ensure that. In another detailed embodiment, the present invention ensures that the expression of one or more plant-expressing genes is modified by specifically modifying one or more of the regulatory elements that lead to the expression of the gene. Includes the use of the CRISPR / Cas9 system as described herein.

In a detailed embodiment, the invention includes methods involving the introduction of foreign genes and / or the targeting of endogenous genes and their regulatory elements, such as those listed below.

1. 1. Genes that confer resistance to pests or diseases:
-Plant disease resistance gene. By transforming a plant with a clone resistance gene, it is possible to engineer a plant that is resistant to a specific pathogen strain. For example, Jones et al. , Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulpum); Martin et al. , Science 262: 1432 (1993) (Tomato Pto gene for resistance to Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinos et al. , Cell 78: 1089 (1994) (Arabidops can be the RSP2 gene for resistance to Pseudomonas syringae)).
-A gene that imparts resistance to pests such as soybean cyst nematodes. See, for example, PCT Application International Publication No. 96/30517 Pamphlet; PCT Application International Publication No. 93/19181 Pamphlet.
Bacillus thuringiensis proteins, such as Geisser et al. , Gene 48: 109 (1986).
Lectins, such as Van Damme et al. , Plant Molec. Biol. 24:25 (see 1994).
Vitamin-binding proteins such as avidin, see PCT Application US93 / 06487, teaching the use of avidin and avidin homologs as larval eradication agents against pests.
-Enzyme inhibitors such as proteases or proteinase inhibitors or amylase inhibitors. For example, Abe et al. , J. Biol. Chem. 262: 16793 (1987), Huub et al. , Plant Molec. Biol. 21: 985 (1993)), Sumitani et al. , Biosci. Biotechnology. Biochem. 57: 1243 (1993) and US Pat. No. 5,494,813.
-Insect-specific hormones or pheromones such as ecdysteroids or juvenile hormones, variants thereof, mimetics based on them, or antagonists or agonists thereof. For example, Hammock et al. , Nature 344: 458 (1990).
-Insect-specific peptides or neuropeptides that, upon expression, disrupt the physiological function of the pest of the disease source. For example, Regan, J. et al. Biol. Chem. 269: 9 (1994) and Pratt et al. , Biochem. Biophyss. Res. Comm. 163: 1243 (1989). See also U.S. Pat. No. 5,266,317.
An insect-specific venom naturally produced by snakes, wasps, or any other organism. For example, Pang et al. , Gene 116: 165 (1992).
Enzymes involved in the excessive accumulation of monoterpenes, sesquiterpenes, steroids, hydroxamic acids, phenylpropanoid derivatives or other non-protein molecules with insecticidal activity.
Enzymes involved in the modification of biologically active molecules, including post-translational modifications; for example, glycolytic enzymes, proteolytic enzymes, lipolytic enzymes, nucleases, cyclases, transaminase, esterases, hydrolases, phosphatases, kinases, phosphorylases, Polymerases, esterases, chitinases and glucanases (whether natural or synthetic). PCT Application International Publication No. 93/02197 Pamphlet, Kramer et al. , Insect Biochem. Molec. Biol. 23: 691 (1993) and Kawaleck et al. , Plant Molec. Biol. 21: 673 (1993).
-Molecules that stimulate signal transduction. For example, Bottlera et al. , Plant Molec. Biol. 24: 757 (1994), and Griess et al. , Plant Physiol. 104: 1467 (1994).
-A virus-invasive protein or a complex toxin derived from it. Beachy et al. , Ann. rev. Phytopasol. See 28: 451 (1990).
-A development-suppressing protein naturally produced by a pathogen or parasite. Lamb et al. , Bio / Technology 10: 1436 (1992) and Toubart et al. , Plant J. 2: 367 (1992).
-A development-suppressing protein naturally produced by plants. For example, Logemann et al. , Bio / Technology 10: 305 (1992).
• In plants, pathogens are often host-specific. For example, one Fusarium species can cause tomato wilt, but only tomatoes attack, while other Fusarium species attack only wheat. Plants have existing induced defenses and resist many pathogens. Mutation and recombination events between plant generations result in susceptibility to genetic variation, especially as pathogens replicate more frequently than plants. Plants may also have non-host resistance, such as partial resistance to any pathogen strain, for example, where the host is incompatible with the pathogen or is typically regulated by many genes, and / or. There may also be complete resistance to some pathogen strains that are not present to other strains. Such resistance is typically regulated by several genes. By using the methods and components of the CRISPR-Cas9 system, there are novel tools here to proactively induce specific mutations. Therefore, the production of resistance genes can be induced by analyzing the genome of the source of the resistance gene and using the CRISPR / Cas9 system methods and components in plants with the desired properties or traits. .. This system can do it with higher accuracy than previous mutagens, which in turn can accelerate and improve plant breeding programs.

2. 2. Genes involved in plant diseases, such as those listed in International Publication No. 201346247 Pamphlet:
-Rice disease: Rice blast fungus (Magnaporthe grisea), rice sesame leaf blight fungus (Cochliobolus miyabeanus), rice crest blight fungus (Rhizoctonia solani), rice blight fungus (Fusarium head blight), Fusarium head blight (Fusarium head blight), Fusarium head blight (Fusarium head blight) (Erysiphy gramnis), Fusarium head blight (Fusarium head blight), Fusarium head blight (F. avenaceum), Fusarium head blight (F. culmorum), Fusarium head blight (Microdochium nivari), Fusarium head blight (Fusarium head blight), Fusarium head blight (Fusarium head blight), Fusarium head blight (Fusarium head blight) ), Barley Fusarium head blight (P. gramnis), Barley Fusarium head blight (P. recondita), Barley red snow rot (Micronectriera nivale), Barley snow rot small granule nuclear disease (Typula sp.), Barley Fusarium head blight (Ustyla) tritici), Fusarium head blight (Tilletia carriers), Fusarium head blight (Pseudocercosporella herpotorichoides), Fusarium head blight (Mycosphaerella fusarium head blight), Fusarium head blight (Fusarium head blight), Fusarium head blight (Fusarium head blight), Fusarium head blight (Pseudocercosporella herpotorichoides), Fusarium head blight (Mycosphaerella fusarium) Diseases: Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight, Fusarium head blight. Fusarium head blight (Puccinia striiformis), Fusarium head blight (P. graminis), Fusarium head blight (P. hordei), Fusarium head blight (Ustilago fusarium head blight), Fusarium head blight (Ustilago fusarium head blight), Fusarium head blight (R) (Pyrenophora teres), Barley spot fungus (Cochliobolus sativus), Barley spot fungus (Pyrenophora graninea), Barley blight fungus (Rhizoctonia solani) Diseases of corn: Corn smut bacterium (Ustilago maydis), corn sesame leaf blight fungus (Cochliobolus heterostrophus), corn corn smut fungus (Gloeocercospora sorghi), corn southern rust fungus (Puccinea corn scab) ), Corn smut (Rhizoctonia solani);
・ Diseases of citrus fruits: Phytophthora citrus, Phytophthora phytophthora, Phytophthora phytophthora, Phytophthora phytophthora, Phytophthora phytophthora, phytophthora phytophthora, phytophthora phytophthora Phytophthora citropthora; Diseases of apples: Phytophthora phytophthora, Phytophthora phytophthora, Phytophthora phytophthora, Phytophthora phytophthora phytophthora phytophthora phytophthora phytophthora phytophthora phytophthora phytophthora phytophthora (Venturia inaequalis), Phytophthora catetum, Phytophthora bactorum;
・ Diseases of pear: Pear scab (Venturia nashicola), Pear scab (V. pilina), Pear scab (Alternaria alternata Japanacee pear pear potato), Pear scab (Pear spore), Pear scab (Pear spore) );
Peach disease: Monilinia fructicola, Cladosporium carpophilum, Phomopsis sp.;
-Grape disease: Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica, Plasmopara vitica Plasmopara viticola;
・ Diseases of oysters: Persimmon anthracnose (Gloesporium kaki), Persimmon keratoses (Cercospora kaki), Persimmon round star foliage (Mycosphaerela nawae);
・ Diseases of melons: Colletotrichum lagenaria, phytophthora phytophthora, phytophthora melonis, phytophthora melonis, melons, melons, melons, melons, melons, melons, melons, melons, melons (Pseudoperonospora cubensis), Phytophthora sp., Phytophthora sp., Pythium sp.
-Tomato diseases: Tomato ring-spot fungus (Alternaria solani), tomato leaf mold fungus (Cladosporium fullvum), tomato epidemic fungus (Phytophthora influenza);
・ Eggplant disease: Phomopsis vexans, Erysiphe cichoracearum;
Diseases of cruciferous vegetables: cruciferous plants Black spot fungus (Alternaria japonica), cruciferous plant white spot disease fungus (Cercosporella brassicae), cruciferous plant root-knot disease fungus (Plasmodiophora brassicae), cruciferous plants (Plasmodiophora brassicae), cruciferous plants
・ Welsh onion disease: Leek rust (Puccinia alli), Welsh onion disease (Peronospora destructor);
-Soybean diseases: Soybean spot fungus (Cercospora kikuchii), Soybean black spot fungus (Elsinoe glycines), Soybean black spot fungus (Diaporthe phaseolorum var. Sojae), Soybean brown spot fungus (Septoria sp.) Soybean spot fungus (Septoria gycines) Soybean rust fungus (Phakoposora pachyrhizi), soybean stalk epidemic fungus (Phytophthora sojae), soybean lysoctonia root rot fungus (Rhizoctonia solani), soybean brown ring spot fungus (Corynespora soybean) soybean fungus (Corynespora soybean)
・ Diseases of common bean: Common bean anthrax (Colletrichum lindemthianum);
Peanut diseases: peanut black astringent fungus (Cercospora personata), peanut brown spot fungus (Cercospora arachidicola), peanut white silk disease fungus (Sclerotium lfsiii);
・ Peas disease Peas: Peas powdery mildew (Erysiphe pisi);
-Potato disease: Alternaria solani, Phytophthora infestans, Phytophthora erythroseptica, Potato powder scorpion, Potato solani, Potato solani, Potato solani, Potato solani, Potato solani.
-Strawberry disease: Strawberry powdery mildew (Strawberry powdery mildew), Strawberry anthrax (Glomerella cingulata);
・ Tea plant diseases: Tea plant blast fungus (Exobasideium reticularatum), Tea white spot disease fungus (Elsinoe leucospila), Tea ring spot fungus (Pestalotiopsis sp.), Tea anthrax fungus (Colletorichutum sea)
-Tobacco diseases: Tobacco red scab (Alternaria lingipes), Tobacco phytophthora (Erysipe cichoracearum), Tobacco charcoal bacillus (Colletotrichum tabacum), Tobacco downy mildew (Peronospora tobacco)
-Rhizoctonia rape: Sclerotinia sclerotiorum, Rhizoctonia solani;
-Cotton disease: Cotton hip fold disease (Rhizoctonia solani);
・ Diseases of sugar beet: Cercospora beticola, Rhizoctonia chinensis (Thanatephorus cucumeris), Rhizoctonia chinensis (Thanatephorus cucumeris), Aphanomyces cocolis
-Rose disease: Diplocarpon rosae, Sphaerotheca pannosa, Downy mildew (Peronospora sprasa);
Diseases of chrysanthemums and plants of the family Asteraceae: Bremia lactuca, Septria chrysanthemi-indici, Puccinia horiana;
・ Diseases of various plants: Pythium aphanidermatum, Pythium debarianum, Pythium graminicola, Phythium primiulium, Pythium primulium, Pythium cinerea, Pythium cinerea, Pythium cinerea , Botrytis cinerea, Sclerotinia sclerotiorum;
・ Radish disease: Radish black spot fungus (Alternaria brassicola);
-Shiva disease: Shiva dollar spot disease fungus (Sclerotinia homeocarpa), Shiva leaf rot fungus (Rhizoctonia solani);
-Banana diseases: Banana black sigatoka (Mycosphaerella fijiensis), Banana spot leaf disease (Mycosphaerella mycosphaerella);
・ Sunflower disease: Sunflower beetle (Plasmopara halstedi);
-Aspergillus spp., Penicillium spp., Fusarium spp., Gibberella spp., Trichoderma spp., Trichoderma spp., Tricoderma spp. Gibberella spp.), Rhizopus spp., Rhizopus spp., Mucor spp., Corticium spp., Roma spp., Rhoma spp., Rhizopus spp. Diseases of seeds of various plants caused by species of the genus Gibberella (Diplodia spp.) Or diseases in the early stage of growth;
-Viral diseases of various plants transmitted by species of the genus Polymixa (Polymixa spp.), Species of the genus Olipidium (Olpidium spp.), Etc.

3. 3. Examples of genes that confer herbicide resistance:
Resistance to herbicides that inhibit growth points or meristems, eg, Lee et al. , EMBO J. 7: 1241 (1988), and Miki et al. , Theor. Apple. Genet. 80: 449 (1990), such as imidazolinone or sulfonylurea.
Glyphosate resistance (eg, resistance conferred by the mutant 5-enolpylvirsimimic acid-3-phosphate synthase (EPSP) gene, aroA gene and glyphosate acetyltransferase (GAT) gene, respectively), or by gluhosinate. Resistance to other phosphono compounds such as (Streptomyces hygroscopicus and Streptomyces viridichromogenes), including Streptomyces viridichromogenes, Streptomyces genus (Streptomyces) transferase from the genus Streptomyces (Streptomyces) Gene), and resistance to pyridinoxy or phenoxypropionic acid and cyclohexone by the ACCase inhibitor coding gene. For example, US Pat. Nos. 4,940,835 and US Pat. No. 6,248,876, US Pat. No. 4,769,061 and EP0 333:033 and US Pat. No. 4, See 975, 374. In addition, EP0242246, DeGreen et al. , Bio / Technology 7:61 (1989), Marshall et al. , Theor. Apple. Genet. 83: 435 (1992), Castle et. al. See also International Publication No. 2005012515 Pamphlet and International Publication No. 2005107437 Pamphlet.
・ Przivilla et al. , Plant Cell 3: 169 (1991), US Pat. No. 4,810,648, and Hayes et al. , Biochem. J. Herbicide resistance that inhibits photosynthesis, such as triazine (psbA and gs + genes) or benzonitrile (nitrilase gene) in 285: 173 (1992), and glutathione S-transferase.
A gene encoding a mutant glutamine synthetase enzyme that is resistant to an enzyme or inhibition that detoxifies herbicides, such as US Patent Application No. 11 / 760,602. Alternatively, the detoxifying enzyme is an enzyme encoding a phosphinotricine acetyltransferase (such as a bar or pat protein from the genus Streptomyces). Regarding phosphinotricine acetyltransferase, for example, US Pat. Nos. 5,561,236; 5,648,477; 5,646,024; 5,273, 894; 5,637,489; 5,276,268; 5,739,082; 5,908,810 and the same. No. 7,112,665.
Hydrohydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, i.e., naturally occurring HPPD-resistant enzymes, or WO 96/38567, Pamphlet 99/24585, and Pamphlet 99/24586. A gene encoding a mutation or chimeric HPPD enzyme as described in Pamphlet, WO 2009/144079, Pamphlet 2002/046387, or US Pat. No. 6,768,044.

4. Examples of genes involved in abiological stress tolerance:
Reduced expression and / or activity of the poly (ADP-ribose) polymerase (PARP) gene in plant cells or plants as described in International Publication No. 00/04173 or International Publication No. 2006/045633. A trans gene that can be made to.
A transgene capable of reducing the expression and / or activity of a PARG-encoding gene in a plant or plant cell, as described, for example, in International Publication No. 2004/090140 Pamphlet.
-For example, as described in EP04077624.7, International Publication No. 2006/133827, PCT / EP07 / 002,433, EP1999263, or International Publication No. 2007/10732. , Nicotinamide adenine dinucleotide salvage synthetic pathway, including nicotinamide adenine transferase, nicotinic acid mononucleotide adenyl transferase, nicotinamide adenine dinucleotide synthetase or nicotinamide phosphoribosyl transferase. A trans gene that encodes an enzyme.
-As enzymes involved in carbohydrate biosynthesis, for example, EP0571427, International Publication No. 95/04826, EP0719338, International Publication No. 96/15248, International Publication No. 96/19581, International. Publication No. 96/27674 Pamphlet, International Publication No. 97/11188 Pamphlet, International Publication No. 97/26362 Pamphlet, International Publication No. 97/32985 Pamphlet, International Publication No. 97/4328 Pamphlet, International Publication No. 97/44472 No. Pamphlet, International Publication No. 97/45545, International Publication No. 98/27212 Pamphlet, International Publication No. 98/40503 Pamphlet, International Publication No. 99/58688 Pamphlet, International Publication No. 99/58690 Pamphlet, International Publication 99/58654 Pamphlet, International Publication 00/08184 Pamphlet, International Publication 00/08185 Pamphlet, International Publication 00/08175 Pamphlet, International Publication 00/28052 Pamphlet, International Publication 00/7729 Pamphlet, International Publication No. 01/12782 Pamphlet, International Publication No. 01/12826 Pamphlet, International Publication No. 02/101059 Pamphlet, International Publication No. 03/071860 Pamphlet, International Publication No. 2004/056999 Pamphlet, International Publication No. 2005/030942 Pamphlet, International Publication No. 2005/030941 Pamphlet, International Publication No. 2005/0956332 Pamphlet, International Publication No. 2005/095617 Pamphlet, International Publication No. 2005/095619 Pamphlet, International Publication No. 2005/095618 Pamphlet , International Publication No. 2005/1293927 Pamphlet, International Publication No. 2006/018319 Pamphlet, International Publication No. 2006/103107 Pamphlet, International Publication No. 2006/108702 Pamphlet, International Publication No. 2007/090823 Pamphlet, International Publication No. 00 / 22140 Pamphlet, International Publication No. 2006/063862 Pamphlet, International Publication No. 2006/07263 Pamphlet, International Publication No. 02/034923 Pamphlet, EP06090134.5, EP06090228.5, EP06090227.7 Specification, EP07090007.1. Specification, EP0709009.7, International Publication No. 01/14569 Pamphlet, International Publication No. 02/79410 Pamphlet, International Publication No. 03/33540 Pamphlet, International Publication No. 2004/079883 Pamphlet, International Publication No. 01 / 19975 Pamphlet, International Publication 95/26407 Pamphlet, International Publication 96/34968 Pamphlet, International Publication 98/20145 Pamphlet, International Publication 99/12950 Pamphlet, International Publication 99/6605 Pamphlet, International Publication No. 99/53072 Pamphlet, US Patent No. 6,734,341, International Publication No. 00/11192 Pamphlet, International Publication No. 98/22604 Pamphlet, International Publication No. 98/32326 Pamphlet, International Publication 01/98509 Pamphlet, International Publication 01/98509 Pamphlet, International Publication No. 2005/002359 Pamphlet, US Pat. No. 5,824,790, US Pat. No. 6,013,861, International Published in Publication No. 94/04693 Pamphlet, International Publication No. 94/09144, International Publication No. 94/11520, International Publication No. 95/35026 or International Publication No. 97/20936, or Polyfluctose as disclosed in EP 0663956, International Publication 96/01904, International Publication 96/21023, International Publication 98/39460, and International Publication 99/24593. , In particular the production of inulin and levan-type polyfluctose, International Publication No. 95/31553, US Patent Application Publication No. 2002031826, US Patent Nos. 6,284,479, US Patent No. 5,712. Α-1, as disclosed in Specification 107, International Publication No. 97/47806 Pamphlet, International Publication No. 97/47807 Pamphlet, International Publication No. 97/47808 Pamphlet and International Publication No. 00/14249 Pamphlet, Production of 4-glucans, production of α-1,6-branched α-1,4-glucan as disclosed in Pamphlet 00/73422, International Publication No. 00/47727, International Publication No. 00/47727. Published 00/73422 Pamphlet, EP06077301 . Production of alternan as disclosed in Specification 7, US Pat. Nos. 5,908,975 and EP0728213, eg, WO 2006/032538 Pamphlet, WO 2007/039314. Pamphlets, International Publication No. 2007/039315 Pamphlet, International Publication No. 2007/039316 Pamphlet, Japanese Patent Application Laid-Open No. 2006-304779, and International Publication No. 2005/012529 Pamphlet contains enzymes involved in hyaluronan production. Can be mentioned.
-A gene that improves drought resistance. For example, WO 2013122472 states that the absence or level of the functional ubiquitin protein ligase protein (UPL) protein, more specifically the UPL3, reduces the water requirements of the plant or improves its drought resistance. It is disclosed that it leads to. Other examples of transgenic plants with increased drought tolerance are described, for example, in U.S. Patent Application Publication No. 2009/014485, U.S. Patent Application Publication No. 2007/02666453, and WO 2002/083911. It is disclosed. U.S. Patent Application Publication No. 2009/01448550 describes plants that exhibit a drought-tolerant phenotype due to altered expression of the DR02 nucleic acid. U.S. Patent Application Publication No. 2007/02666453 describes plants exhibiting a drought-tolerant phenotype due to altered expression of DR03 nucleic acids, and WO 2002/083911 is expressed in guard cells. Describes plants that have increased resistance to drought stress due to reduced activity of their ABC transporters. Another example is a study by Kasuga and co-authors (1999), in which overexpression of the cDNA encoding DREB1A in transgenic plants activates the expression of many stress tolerance genes under normal growth conditions. It is described that it has improved drought resistance, salt load resistance, and freezing resistance. However, expression of DREB1A also resulted in significant growth retardation under normal growth conditions (Kasuga (1999) Nat Biotechnol 17 (3) 287-291).

In a more detailed embodiment, the crop plant can be improved by influencing a particular plant trait. For example, development of pesticide-resistant plants, improvement of disease resistance in plants, improvement of plant insect and nematode resistance, improvement of plant resistance to parasitic weeds, improvement of plant drought resistance, improvement of plant nutritional value. Improvement, improvement of plant stress tolerance, avoidance of self-pollution, plant feed digestible biomass, grain yield, etc. Some specific non-limiting examples are provided herein below.

In addition to single-gene targeted mutations, the Cas9CRISPR complex includes targeted mutations of multiple genes in plants, deletion of chromosomal fragments, site-specific integration of transgenes, site-specific mutagenesis in vivo, And can be designed to allow accurate gene substitution or allogeneic swapping. Therefore, the methods described herein have wide applicability in gene discovery and validation, mutation and cisgenic breeding, and hybrid breeding. These applications promote the production of new generation genetically modified crops with a variety of improved agricultural traits, including herbicide resistance, disease resistance, abiological stress resistance, high yields, and high quality.

Use of the Cas9 gene to produce male sterile plants Hybrid plants typically have favorable agricultural traits compared to pure plants. However, for self-pollinating plants, hybrid production can be challenging. In various plant types, genes important for plant fertility, more specifically male fertility, have been identified. For example, in maize, at least two genes important in fertility have been identified (Amitabh Mohanty International Convention on New Plant Breeding Molecule Technology Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Technology, Development, Technology, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Technology, Development, Plant Physiol. 2015 Oct; 169 (2): 931-45; Djukanovic et al. Plant J. 2013 Dec; 76 (5): 888-99). The methods provided herein can be used to target the genes required for male fertility to produce male sterile plants that can be easily crossed to produce hybrids. In a detailed embodiment, the CRISPR / Cas9 system provided herein is used to induce targeted mutagenesis of the cytochrome P450-like gene (MS26) or meganuclease gene (MS45), thereby causing maize sterility in maize plants. Granted. Such genetically altered maize plants can be used in hybrid breeding programs.

Increasing fertility in plants In a detailed embodiment, the fertility of plants, such as rice plants, is extended using the methods provided herein. For example, by targeting a rice fertile phase gene such as Ehd3, mutations can be generated in that gene, and small plants can be selected for the extended regenerated plant fertile phase (CN 104004782). As stated in the book)

Use of Cas9 to generate genetic variation in crops of interest Wild fertility availability and genetic variation in crop plants are key to crop improvement programs, but the variety of available fertility from crop plants Gender is limited. The present invention envisions a method of producing a variety of genetic variation in a germ of interest. This application of the CRISPR / Cas9 system provides a library of chi / sgRNAs that target various locations within the plant genome and is introduced into plant cells along with the Cas9 effector protein. In this way, a collection of genome-wide point mutations and gene knockouts can be generated. In a detailed embodiment, the method comprises the step of producing a plant site or plant from the cells so obtained, and the step of screening the cells for the trait of interest. The target gene can contain both coding and non-coding regions. In a detailed embodiment, the trait is stress resistant and the method is a method of producing stress resistant crop varieties.

Use of Cas9 to Affect Fruit Ripeness Ripeness is a normal step in the fruit and vegetable ripening process. Once ripening begins, it makes the fruit or vegetable unsuitable for eating in just a few days. This process causes significant losses to both farmers and consumers. In a detailed embodiment, the methods of the invention are used to reduce ethylene production. This is ensured by ensuring one or more of the following: a. Suppression of ACC synthase gene expression. ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the enzyme involved in the conversion of S-adenosylmethionine (SAM) to ACC; the second to last step in ethylene biosynthesis. Insertion of an antisense (“mirror image”) or truncated copy of the synthase gene into the plant genome interferes with enzyme expression; b. Insertion of the ACC deaminase gene. The gene encoding this enzyme is obtained from the common non-pathogenic soil bacterium Pseudomonas chlorolaphis. This converts the ACC into another compound, thereby reducing the amount of ACC available for ethylene production; c. Insertion of SAM hydrolase gene. This procedure is similar to ACC deaminase, where reducing the amount of its metabolite precursor interferes with ethylene production; in this case SAM is converted to homoserine. The gene encoding this enzyme is obtained from E. coli T3 bacteriophage, and d. Suppression of ACC oxidase gene expression. ACC oxidase is an enzyme that catalyzes the oxidation of ACC to ethylene, the final step in the ethylene biosynthetic pathway. Downregulation of the ACC oxidase gene using the methods described herein results in suppression of ethylene production, which delays fruit ripening. In a detailed embodiment, in addition to or in lieu of the modifications described above, the methods described herein are used to modify ethylene receptors, thereby interfering with the ethylene signal obtained by the fruit. In a detailed embodiment, the expression of the ETR1 gene encoding an ethylene binding protein is modified and more specifically suppressed. In a detailed embodiment, in addition to or in lieu of the modifications described above, the methods described herein are polygalacturo, an enzyme involved in the degradation of the substance pectin that maintains the integrity of the plant cell wall. It is used to modify the expression of a gene encoding an enzyme (PG). Pectin degradation occurs at the beginning of the ripening process, resulting in fruit softening. Therefore, in a detailed embodiment, the production of the PG enzyme is reduced by introducing a mutation into the PG gene using the method described herein or by suppressing the activation of the PG gene. This delays pectin degradation.

Thus, in a detailed embodiment, the method comprises using a CRISPR / Cas9 system to ensure one or more alterations of the plant cell genome, such as those described above, and regenerating the plant from it. .. In a detailed embodiment, the plant is a tomato plant.

Increasing the shelf life of a plant In a detailed embodiment, the methods of the invention are used to modify genes involved in the production of compounds that affect the shelf life of a plant or plant site. More specifically, the modification is a modification in a gene that prevents the accumulation of reducing sugars in potato tubers. When subjected to high temperature treatment, these reducing sugars react with free amino acids to produce a brown bitter product, and the level of acrylamide, which is a potential carcinogen, rises. In a detailed embodiment, the methods provided herein reduce or inhibit the expression of the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose into glucose and fructose (encoding). Clasen et al. DOI: 10.1111 / pbi.12370).

Use of the CRISPR / Cas9 system to ensure value-added traits In a detailed embodiment, the CRISPR / Cas9 system is used to produce nutritionally improved agricultural crops. In a detailed embodiment, the methods provided herein are "functional foods," i.e. modified foods or food ingredients that may provide health benefits beyond the conventional nutrients they contain, and / Or "nutrienticals", ie foods or adapted to produce substances that can be considered as part of a food product and provide health benefits, including prevention and treatment of disease. In a detailed embodiment, Neutraluticals are useful for the prevention and / or treatment of one or more of cancer, diabetes, cardiovascular disease, and hypertension.

Examples of nutritionally improved crops include: (Newell-McGlouglin, Plant Physiology, July 2008, Vol. 147, pp. 939-953):
-Bahiagrass (Luciani et al. 2005, Florida Genetics Context Poster), Canola (Roesler et al., 1997, Plant Physiol 113 75-81), Corn (Cromwell et al, 1967, 1967, 1969) 'Quin et al. 2000 J Anim Sci 78 2144-2149, Yang et al. 2002, Transgenic Res 11 11-20, Young et al. 2004, Plant J 38 910-922), Potato (Yu J and Ao, 1997) Bot Sin 39 329-334; Chakraborty et al. 2000, Proc Natl Acad Sci USA 97 3724-3729; Li et al. 2001) Chin Sci Bull 46 482-484, Rice (Katsube19) 1074), soybeans (Dinkins et al. 20011, Rapp 2002, In Vitro Cell Dev Biol Plant 37 742-747), sweet potatoes (Egnin and Prakash 1997, In Vitro Cell Dev Biol 33, etc.) Protein quality, content and / or amino acid composition.
-Canola (Falco et al. 1995, Bio / Technology 13 577-582), Lupinus (White et al. 20011, J Sci Food Agric 81 147-154), Corn (Lai and Massing, 2002 Ag). March 11 and 2008)), potatoes (Zheh et al. 20011, Plant Physiol 127 792-802), sorghum (Zhao et al. 2003, Klewer Academic Publics, Dordrecht, The Felce, The 16th) Essential amino content, such as that described for al. 1995 Bio / Technology 13 577-582; Galilli et al. 2002 Crit Rev Plant Sci 21 167-204).
・ Canola (Dehesh et al. (1996) Plant J 9 167-172 [PubMed]; Del Veccio (1996) INFORMATION News on Fats, Oils and Related Materials 1916.liters7 230-243; 75-81 [PMC free article] [PubMed]; Froman and Ursin (2002, 2003) Abstracts of Papers of the American Chemical Society Chemical Society 223 U35; James-1 Agbios (2008, above); Wata (Chapman et al. (2001). J Am Oil Chem Soc 78 941-147; Liu et al. (2002) J Am Coll Nutr 21 205S-211S [PubMed]; O'N 2007) American Life Plantist.http: //www.biotechnews.com.au/index.php/id; 866694817; fp; 4; fpid; 2 (June 17, 2008)), Linda, et al. Plant Cell 16: 2734-2748), Corn (Young et al., 2004, Plant J 38 910-922), Oil palm (Jalani et al. 1997, JA Am Oil Chem Soc 74 1451-1455; Parveez, 2003, AgBi 113 1-8), rice (Ani et al., 2003, Plant Cell Rep 21 988-992), soybeans (Reddy and Thomas, 1996, Nat Biotechnol 14 639-642; Kinney and Kwolton, 1998, Bolton, 1998). Oils and fatty acids, such as those related to London, pp 193-213), sunflowers (Arcadaia, Biosciences 2008), etc. Kori (Smeekens (1997) Trends Plant Sci 2 286-287, Springer et al. (1997) FEBS Lett 400 355-358, Sevenier et al. (1998) Nat Biotechnol 16 843-846), corn (Caimi et al. (1996) Plant Physiol 110 355-363), potatoes (Hellwege et al., 1997 Plant J 12 1057-1065), sugar beet. 1997, described above), inulin (Hellewege et al. 2000, Proc Nature Acad Sci USA 97 8699-8704), described with respect to potatoes, starch (Schwall et al.) Described with respect to rice. (2000) Nat Biotechnol 18 551-554, Chain et al. (2005) Mol Breed 15 125-143), etc.
-Canola (Shintani and DellaPenna (1998) Science 282 2098-2100), Corn (Rocheford et al. (2002). J Am Coll Nutr 21 191S-198S, Cahon et al. (2003) 108T. et al. (2003) Proc Natl Acad Sci USA 100 3525-3530), corn seeds (Shewmaker et al. (1999) Plant J 20 401-412, potatoes (Ducreux et al., 2005, J Exp 81-56) ), Rice (Ye et al. (2000) Science 287 303-305, Strawberry (Agius et al. (2003), Nat Biotechnol 21 177-181), Tomato (Rosati et al. (2000) Plant J 24 413-419) , Fraser et al. (2001) J Sci Food Agric 81 822-827, Mehta et al. (2002) Nat Biotechnol 20 613-618, Diaz de la Garza et al. (2004) Proc1A c1 , Enfissi et al. (2005) Plant Biotechnol J 3 17-27, Della Penna (2007) Proc Natl Acad Sci USA 104 3675-3676 and other vitamins and carotenoids.
-Apples (Stilbenes, Szankouski et al. (2003) Plant Cell Rep 22: 141-149), Alfalfa (Resveratrol, Hipskind and Paiva (2000) Mol Plant Microbe Interact 13 551-562), Keywi-les , Kobayashi et al. (2000) Plant Cell Rep 19 904-910), corn and soybeans (flavonoids, Yu et al. (2000) Plant Physiol 124 781-794), potatoes (anthocyanin and alkaloid glycosides, Lukaszewiz. (2004) J Agric Food Chem 52 1526-1533), rice (flavonoids and resveratrol, Stark-Lorenzen et al. (1997) Plant Cell Rep 16 668-673, Shin et al. (2006) Plant Bio 303-315), tomatoes (+ resveratrol, chlorogenic acid, flavonoids, stilbene; Rosati et al. (2000) above, Mir et al. (2001) Nature 19 470-474, Nigeweg et al. (2004) Nat Functionality described with respect to Biotechnol 22 746-754, Giovinazzo et al. (2005) Plant Biotechnol J 3 57-69), apples (coffee and ferulic acid, resveratrol; United Press International (2002)). Next metabolites; and alfalfa (fitase, Austin-Phillips et al. (1999) http: // www.molecularfarming.com/nonmedical.html), lettuse (iron, Goto et al. (2000) Theor. Genet 100 658-664), rice (iron, Lucca et al. (2002) JAm Coll Nutr 21 184S-190S), corn, soybeans and wheat (whate) (phytase, Drakawaki et al. (200) 5) Plant Mol Biol 59 869-880, Denbow et al. (1998) Port Sci 77 878-881, Brinch-Pedersen et al. (2000) Mineral availability as described with respect to Mol Breed 6 195-206).

In a detailed embodiment, the value-added trait relates to the expected health benefits of the compounds present in the plant. For example, in a detailed embodiment, value-added crops are obtained by applying the methods of the invention to ensure modification of one or more of the following compounds, or to induce / increase their synthesis:
-For carotenoids and green vegetables such as α-carotene present in carrots (neutralizes free radicals that can cause damage to cells) or β-carotene present in various fruits and vegetables (neutralizes free radicals) Existing lutein (contributing to maintaining healthy vision),
Lycopene present in tomatoes and tomato products (believed to reduce the risk of prostate cancer)
・ Zeaxanthin present in citrus fruits and corn (contributes to maintaining healthy eyesight),
Dietary fiber such as insoluble fiber present in wheat bran (which can reduce the risk of breast and / or colon cancer) and soluble fiber present in β-glucan, psyllium and whole grains present in oat wheat (cardiovascular disease (cardiovascular disease) CVD) risk can be reduced)
-Fatty acids such as omega-3 fatty acids (which can reduce the risk of CVD and improve mental and visual function), conjugated linoleic acid (which can improve body composition, can reduce the risk of certain cancers) , And GLA (which can reduce the risk of cancer and CVD inflammation, can improve body composition)
Flavonoids such as hydroxycinnamic acid present in wheat with antioxidant-like activity can reduce the risk of degenerative diseases, flavonols, catechins and tannins present in fruits and vegetables (neutralizing free radicals) And can reduce the risk of cancer)
・ Glucosinolates, indoles, isothiocyanates such as sulforaphane, which are present in cruciferous vegetables (broccoli, kale) and horseradish (can neutralize free radicals and reduce the risk of cancer)
-Phenols such as stillbens present in grapes (which can reduce the risk of degenerative diseases, heart disease, and cancer, can have a longevity effect) and coffee acid and ferulic acid (antioxidants) present in vegetables and citrus fruits. (Has similar activity), can reduce the risk of degenerative, heart and eye diseases, and epicatechin present in cacao (has antioxidant-like activity, can reduce the risk of degenerative and heart disease)
-Plant stannole / sterols present in corn, soybean, wheat and wood-derived oils (lowering blood cholesterol levels can reduce the risk of coronary heart disease)
Fructans, inulins, and fructo-oligosaccharides present in Jerusalem artichoke, shallot, and onion powder (which can improve gastrointestinal health)
・ Saponins present in soybeans (which can lower LDL cholesterol)
-Soy protein present in soybeans (which can reduce the risk of heart disease)
Phytoestrogens such as isoflavones present in soybeans (which can reduce menopausal symptoms such as rash, osteoporosis and CVD can be reduced) and lignans present in flax, limewood and vegetables (heart disease and some cancers) Can protect against LDL cholesterol, can lower total cholesterol).
-Sulfides and thiols such as diallyl sulfide present in onions, garlic, olives, sardines and scallons, and allylmethyltrisulfide and dithiolthiones present in cruciferous vegetables (which can lower LDL cholesterol, health) Helps maintain a healthy immune system)
-Tannins such as proanthocyanidins present in cranberries and cocoa (which can improve urinary tract health and reduce the risk of CVD and hypertension)
·etc.

In addition, the methods of the invention also envision modifying protein / starch function, shelf life, taste / beauty, fiber quality, and allergen, antinutrient, and toxin-reducing traits.

In certain embodiments, the plant may be a legume. The present invention may utilize the CRISPR-Cas9 system disclosed herein, for example, to investigate and modify soybeans, peas, and peanuts without limitation. Curtin et al. Provides a toolbox for legume functional genomics (Curtin et al., "Genome engineering toolbox for legume Functional genomics", International. See Genome Conference XXII 2014). Curtin used CRISPR gene transformation to knock out / knock down a single copy to replicate legume genes in both hairy roots and whole plant lines. Some of the target genes have been selected to explore and optimize the characteristics of knockout / knockdown systems (eg, phytoendesaturase), while others have with Arabidopsis dicer-like genes. It was identified by soybean homology or by genome-wide association analysis of root grain formation in the genus Medicago.

Peanut allergies and, in general, allergies to legumes are serious health problems that actually exist. Using the CRISPR-Cas9 effector protein system of the present invention, the gene encoding the allergen protein of such legumes can be identified and then edited or silenced. Without limitation on such genes and proteins, Nicolaou et al. Has identified allergen proteins for peanuts, soybeans, lentils, peas, lupines, sayamame, and yaenari. Nicolaou et al. , Current Opinion in Allergy and Clinical Immunology 2011; 11 (3): 222).

Accordingly, the present invention includes a method of producing a plant with nutritional value, the method described herein of a gene encoding an enzyme involved in the production of a component of nutritional value. The plant is characterized by increased expression of the constituents of nutritional value, comprising the step of introducing into a plant cell using the CRISPR / Cas9 system as described above, and the step of regenerating the plant from the plant cell. .. In a detailed embodiment, the CRISPR / Cas9 system is used to indirectly modify the endogenous synthesis of these compounds, eg, by modifying one or more transcription factors that control the metabolism of the compounds. Methods for introducing the gene of interest into plant cells using the CRISPR / Cas9 system and / or modifying the endogenous gene are described above herein.

Some specific examples of modifications in plants modified to impart value-added traits are, for example, transforming the plant with the antisense gene of stearyl-ACP desaturase to increase the stearic acid content of the plant. It is a plant whose fatty acid metabolism is modified by. Knultzon et al. , Proc. Natl. Acad. Sci. U.S. S. A. 89: 2624 (1992). Another example is the phytate content by cloning and then reintroducing DNA associated with a single allele that may be involved in a maize mutant characterized by low levels of phytic acid. Is accompanied by a decrease in. See Raboy et al, Maydica 35: 383 (1990).

Similarly, expression of maize (Zea mays) Tfs C1 and R, which regulates the production of flavonoids in the maize aleurone layer under the control of a strong promoter, causes the genus Arabidopsis (Arabidopsis). In thaliana)), activation of the entire pathway probably resulted in a high accumulation rate of anthocyanins (Bruce et al., 2000, Plant Cell 12: 65-80). Della Penna (Welsch et al., 2007 Annu Rev Plant Biol 57: 711-738) found that Tf RAP 2.2 and its interaction partner SINAT2 increased carotene production in the leaves of the genus Arabidopsis. Expression of Tf Dof1 induces upregulation of the gene encoding the carbon skeleton-producing enzyme in the genus Arabidopsis, a marked increase in amino content, and a decrease in Gluc levels (Yanagisawa, 2004 Plant Cell Physiol 45). : 386-391), and DOF Tf AtDof1.1 (OBP2) upregulated all stages of the glucosinolate biosynthetic pathway in the genus Arabidopsis (Skirycz et al., 2006 Plant J 47: 10-). 24).

Reducing Allergens in Plants In a detailed embodiment, the methods provided herein are used to create plants with reduced allergen levels so that the plants are safer for consumers. In a detailed embodiment, the method comprises modifying the expression of one or more genes involved in the production of plant allergens. For example, in a detailed embodiment, the method downregulates the expression of the Lol p5 gene in a plant cell, such as a ryegrass plant cell, and removes the plant from those plant cells to reduce the pollen allergenicity of the plant. Includes steps to regenerate (Bhalla et al. 1999, Proc. Natl. Acad. Sci. USA Vol. 96: 11676-11680).

Methods for Screening Endogenous Genes of Interest The methods provided herein further include genes for value-encoding enzymes involved in the production of nutritionally value-added components, or generally across species, phylums, and plant kingdoms. Allows the identification of genes that affect agricultural traits. Genes involved in certain nutritional aspects of plants, for example, by selectively targeting genes encoding enzymes of metabolic pathways in plants using the CRISPR / Cas9 system as described herein. Can be identified. Similarly, relevant genes can be identified by selectively targeting genes that can affect desirable agricultural traits. Accordingly, the present invention includes a method of screening a gene encoding an enzyme involved in the production of a compound having a specific nutritional value and / or agricultural trait.

Further application of the CRISPR / Cas9 system in plants and yeast The term "biofuel" used in biofuel production is an alternative fuel made from plants and plant-derived resources as used herein. is there. Renewable biofuels can be extracted from organic matter whose energy has been obtained through the process of carbon fixation, or are made by the use or conversion of biomass. This biomass may be used directly as a biofuel or may be converted to a convenient energy-containing material by thermal conversion, chemical conversion, and biochemical conversion. This biomass conversion can result in fuels in the form of solids, liquids or gases. There are two types of biofuels, bioethanol and biodiesel. Bioethanol is mainly produced by the sugar fermentation process of cellulose (starch), which is mostly derived from corn and sugar cane. Biodiesel, on the other hand, is mainly produced from oil crops such as rapeseed, palm and soybean. Biofuels are mainly used in transportation.

Enhancement of plant properties for biofuel production In a detailed embodiment, as described herein to facilitate access by major hydrolysates for more efficient release of sugars during fermentation. The cell wall properties are altered using this method using the CRISPR / Cas9 system. In a detailed embodiment, the biosynthesis of cellulose and / or lignin is modified. Cellulose is a major component of the cell wall. The biosynthesis of cellulose and lignin is co-regulated. By reducing the proportion of lignin in the plant, the proportion of cellulose can be increased. In a detailed embodiment, the methods described herein are used to downregulate lignin biosynthesis in plants, thereby increasing fermentable sugars. More specifically, as disclosed in WO 200864289 A2, using the methods described herein, 4-coumaric acid 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamon bark. Acid-4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT), caffeate O-methyltransferase (COMT), caffeate oil CoA3-O-methyltransferase (CCoAOMT), ferulic acid 5-hydroxylase (F5H), At least the first selected from the group consisting of cinnamil alcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR), 4-coumaric acid-CoA ligase (4CL), monolignol-lignin specific glycosyltransferase, and aldehyde dehydrogenase (ALDH). The lignin biosynthesis gene of 1 is down-regulated.

In a detailed embodiment, the methods described herein are used to produce plant trout with lower acetic acid levels generated during fermentation (see also WO 201096488). More specifically, mutations are generated in the CaslL homolog using the methods disclosed herein to reduce polysaccharide acetylation.

Modification of Yeast for Biofuel Production In a detailed embodiment, the Cas9 enzyme provided herein is used for the production of bioethanol by recombinant microorganisms. For example, by engineering microorganisms such as yeast with Cas9, biofuels or biopolymers can be made from fermentable sugars, and optionally obtained from agricultural waste as a source of fermentable sugars. It is possible to decompose the lignocellulose derived from the fermented plant. More specifically, the present invention provides a method of introducing an exogenous gene required for biofuel production into a microorganism using the CRISPR / Cas9 complex and / or modifying an endogenous gene that can interfere with biofuel synthesis. To do. More specifically, the method comprises the step of introducing into a microorganism such as yeast one or more nucleotide sequences encoding an enzyme involved in the conversion of pyruvic acid to ethanol or another product of interest. In a detailed embodiment, the method ensures the introduction of one or more enzymes that achieve the degradation of cellulose by microorganisms, such as cellulase. In yet another embodiment, the CRISPR / Cas9 complex is used to modify an endogenous metabolic pathway that competes with the biofuel production pathway.

Thus, in a more detailed embodiment, the microorganisms are modified as follows using the methods described herein:
-At least one heterologous nucleic acid encoding a plant cell wall degrading enzyme is introduced, or the expression of at least one endogenous nucleic acid is increased, so that the microorganism is responsible for the expression of the nucleic acid and the production and secretion of the plant cell wall degrading enzyme. It will be possible;
-Optionally, in combination with at least one heterologous nucleic acid encoding the enzyme that converts acetaldehyde to ethanol, at least one heterologous nucleic acid encoding the enzyme that converts pyruvate to acetaldehyde is introduced or at least one intrinsic cause. Increased expression of sex nucleic acids allows the host cell to express the nucleic acid; and / or-at least one nucleic acid encoding an enzyme in the metabolic pathway in the host cell is modified (where the pathway is pyruvin). A metabolite other than ethanol is produced from acid or acetaldehyde, and the modification results in reduced production of the metabolite), or at least one nucleic acid encoding an inhibitor of the enzyme is introduced.

Modifications of Algae and Plants to Produce Vegetable Oils or Biofuels Transgenic algae or other plants such as Rapeseed are particularly useful in the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). obtain. They can be engineered to express or overexpress high levels of oils or alcohols used in the oil or biofuel industry.

US Pat. No. 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii) species using Cas9. Similar tools can be used to apply the CRISPR / Cas9-based methods described herein to Chlamydomonas species and other algae. In a detailed embodiment, Cas9 and chi / sgRNA are introduced into algae by expression using a vector that expresses Cas9 under the control of a constitutive promoter such as Hsp70A-Rbc S2 or β2-tubulin. The chi / sgRNA will be delivered using a vector containing the T7 promoter. Alternatively, Cas9 mRNA and in vitro transcribed chi / sgRNA may be delivered to algae cells. The electroporation protocol follows the standard recommended protocol of the GeneArt Chlamydomonas Engineering Kit.

Use of Cas9 in the production of microorganisms capable of producing fatty acids In a detailed embodiment, fatty acid esters such as fatty acid methyl esters (“FAME”) and fatty acid ethyl esters (“FAEE”) are used using the methods of the present invention. A genetically engineered microorganism capable of producing fatty acid is produced.

In a detailed embodiment, it is envisioned that the genes involved in modifying the amount and / or quality of lipids produced by algal cells are specifically modified. Genes encoding enzymes involved in the fatty acid synthesis pathway include, for example, acetyl-CoA carboxylase, fatty acid synthase, 3-ketoacyl acyl carrier protein synthase III, glycerol-3-phosphate dehydrogenase (G3PDH), enoyl. Acyl carrier protein reductase (enoyl-ACP-reductase), glycerol-3-phosphate acyl transferase, lysophosphatidic acid acyl transferase or diacylglycerol acyl transferase, phospholipids: diacylglycerol acyl transferase, phosphatidic phosphatase, palmitoyl ( It can encode a fatty acid thioesterase such as palmitoyi) protein thioesterase, or a protein having phospholipidase activity. In a further embodiment, it is envisioned to produce diatoms with increased lipid accumulation. This can be achieved by targeting genes that reduce lipid catabolism. Genes involved in the activation of both triacylglycerol and free fatty acids, as well as genes directly involved in β-oxidation of fatty acids such as acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidase activity and phosphoglucomutase Especially useful for use in the methods of the invention. The Cas9 system and methods described herein can be used to specifically activate such genes in diatoms so that their lipid content is increased.

Typically, the host cell is derived from a carbon source present in the medium, such as alcohol, by expression or overexpression of a gene encoding a thioesterase, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase. It can be engineered to produce fatty acid esters. Thus, using the methods provided herein, microorganisms are modified to overexpress or introduce the thioesterase gene, the gene encoding acyl-CoA synthase, and the gene encoding ester synthase. In a detailed embodiment, the thioesterase gene is selected from tesA,'tesA, tesB, fatB, fatB2, fatB3, fatAl, or fatA. In a detailed embodiment, the gene encoding the acyl-CoA synthase was identified encoding dadDJadK, BH3103, pfl-4354, EAV15023, dadDl, dadD2, RPC_4074, dadDD35, dadDD22, phaa39, or an enzyme having the same properties. Selected from genes. In a detailed embodiment, the genes encoding ester synthase are jojoba (Simmondosia chinasis), Acinetobacter sp. ADP, Arcanivorax borkumensis, Pseudomonas aeruginosa, Pseudomonas aeruginosa. Divactor jadensis (Fundibacter jadensis), Pseudomonas aeruginosa (Arabidopsis thaliana), or Alkaligenes eutrovus, or a synthase / acyl-CoA derived from a variant thereof. In addition to or instead, using the methods provided herein, genes encoding acyl-CoA dehydrogenase, genes encoding outer membrane protein receptors, and genes encoding transcriptional regulators of fatty acid biosynthesis. Expression in at least one of said microorganisms is reduced. In a detailed embodiment, one or more of these genes are inactivated, such as by introducing a mutation. In a detailed embodiment, the gene encoding acyl-CoA dehydrogenase is fadeE. In a detailed embodiment, the gene encoding the transcriptional regulator of fatty acid biosynthesis encodes a DNA transcription repressor, such as fabR.

In addition or instead, the microorganism is modified to reduce expression of at least one or both of the gene encoding pyruvate formic acid lyase, the gene encoding lactate dehydrogenase. In a detailed embodiment, the gene encoding pyruvate formic acid lyase is pflB. In a detailed embodiment, the gene encoding lactate dehydrogenase is IdhA. In a detailed embodiment, one or more of these genes are inactivated, such as by introducing a mutation therein.

In a detailed embodiment, the microorganisms are Escherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus, Synechococcus, Synecocsis, Synecostis, Synecostis ), Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluymicor, Kluicylus ), Mucor, Myceliophtra, Penicillium, Fanerochaete, Pleurotas, Trametes, Chrysocellium ), Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces.

Use of Cas9 in the production of microorganisms capable of producing organic acids The methods provided herein are further used for engineering microorganisms capable of producing organic acids, more specifically from pentose or hexose saccharides. Be done. In a detailed embodiment, the method comprises the step of introducing the exogenous LDH gene into a microorganism. In a detailed embodiment, an endogenous gene encoding a protein involved in an endogenous metabolic pathway that additionally or instead produces a metabolite other than the organic acid of interest and that consumes the organic acid. Increases organic acid production in said microorganisms by inactivating. In a detailed embodiment, this modification ensures that the production of metabolites other than the organic acid of interest is reduced. According to a detailed embodiment, the method deletes and / or inactivates at least one engineered gene in an endogenous pathway in which an organic acid is consumed, or produces a metabolite other than the organic acid of interest. It is used to introduce genes encoding products involved in the sexual pathway. In a detailed embodiment, at least one engineered gene deletion or inactivation is pyruvate decarboxylase (pdc), fumaric acid reductase, alcohol dehydrogenase (adh), acet aldehyde dehydrogenase, phosphoenolpyruvate carboxylase (ppc). ), D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh), one or more genes encoding an enzyme selected from the group consisting of lactate 2-monooxygenase. In a further embodiment, the at least one engineered gene deletion and / or inactivation lies in the endogenous gene (pdc) encoding pyruvate decarboxylase.

In a further embodiment, the microorganism is engineered to produce lactate, and at least one engineered gene deletion and / or inactivation lies in the endogenous gene encoding lactate dehydrogenase. In addition or in lieu, the microorganism comprises at least one engineered gene deletion or inactivation of an endogenous gene encoding a cytochrome-dependent lactate dehydrogenase, such as cytochrome B2-dependent L-lactate dehydrogenase.

Use of Cas9 in Yeast Strain-Used Improved Xylose or Cellobiose Generation In a detailed embodiment, the CRISPR / Cas9 system may be applicable to yeast strain-based selection of improved xylose or cellobiose. Errorprone PCR can be used to amplify one (or more) genes involved in xylose-based or cellobiose-based pathways. Examples of genes involved in the xylose utilization pathway and the cellobiose utilization pathway are, without limitation, Ha, S. et al. J. , Et al. (2011) Proc. Natl. Acad. Sci. USA 108 (2): 504-9 and Galazka, J. Mol. M. , Et al. (2010) Science 330 (6000): 84-6. The resulting double-stranded DNA molecular library, each containing a random mutation in such a gene of choice, may be co-transformed into a yeast strain (eg, S288C) with components of the CRISPR / Cas9 lineage, WO 2015138855. As described in the No. Pamphlet, strains with enhanced xylose or cellobiose utilization can be selected.

Use of Cas9 in the preparation of improved yeast strains used for isoprenoid biosynthesis Tadas Jakocinuas et al. Described the successful application of the multiple CRISPR / Cas9 system to genomic engineering up to five different genomic loci in a single transformation step in Saccharomyces cerevisiae, a brewer's yeast (Saccharomyces cerevisiae). Metabolic Engineering Volume 28, March 2015, Pages 213-222), a high-producing strain of mevalonic acid, which is a key intermediate for the industrially important isoprenoid biosynthetic pathway, has been obtained. In a detailed embodiment, the CRISPR / Cas9 system may be applied in the multiple genome engineering methods as described herein to identify additional high-producing yeast strains used for isoprenoid synthesis.

Use of Cas9 in the Creation of Lactate-Producing Yeast Strains In another embodiment, successful application of the multiple CRISPR / Cas9 system is contemplated. Vratislav Stovicek et al. Similar to (Metabolic Engineering Communications, Volume 2, December 2015, Pages 13-22), improved lactate-producing strains can be designed and obtained in a single transformation event. In a detailed embodiment, the CRISPR / Cas9 system is used to simultaneously insert the heterologous lactate dehydrogenase gene and disrupt the two endogenous genes PDC1 and PDC5.

Further Application of the CRISPR / Cas9 System in Plants In a detailed embodiment, the CRISPR system described herein, and preferably the CRISPR / Cas9 system, can be used to visualize genetic factor dynamics. For example, CRISPR imaging can visualize either repetitive or non-repetitive genomic sequences, report telomere length changes and telomere movements, and monitor locus dynamics throughout the entire cell cycle ( Chen et al., Cell, 2013). These methods can also be applied to plants.

Another application of the CRISPR system described herein, and preferably the CRISPR / Cas9 system, is in vitro and in vivo targeted gene disruption positive selection screening (Malina et al., Genes and Development, 2013). These methods can also be applied to plants.

In a detailed embodiment, custom changes can be introduced into the complex epigenome by fusing an inactive Cas9 endonuclease with a histone-modifying enzyme (Rusk et al., Nature Methods, 2014). These methods can also be applied to plants.

In a detailed embodiment, the CRISPR system described herein, and preferably the CRISPR / Cas9 system, is used to purify specific positions of chromatin to identify related proteins and thus their in transcription. The regulatory role can be elucidated (Waldrip et al., Epigenetics, 2014). These methods can also be applied to plants.

In a detailed embodiment, the present invention is capable of cleaving both viral DNA and RNA and can be used as a therapeutic agent for removing viruses in plant systems. Previous studies in humans include single-strand RNA virus, hepatitis C (A. Prince, et al., Proc. Natl. Acad. Sci, 2015) and double-stranded DNA virus, hepatitis B (V. Ramana,). It demonstrates the successful use of CRISPR in the targeting of et al., Sci. Rep, 2015). These methods can also be adapted to the use of the CRISPR / Cas9 system in plants.

In detailed embodiments, the present invention may be used to alter genomic complexity. In a more detailed embodiment, the CRISPR system described herein, and preferably the CRISPR / Cas9 system, is used to disrupt or alter the number of chromosomes in a haploid plant (chromosomes from one parent). (Contains only) can be produced. Such plants can be induced to cause chromosomal duplication and converted to diploid plants containing only homozygous alleles (Karimi-Ashtiyani et al., PNAS, 2015; Antonio et al., Nucleus, 2014). These methods can also be applied to plants.

In a detailed embodiment, the CRISPR / Cas9 system described herein can be used for self-cleaving. As described, the promoters of the Cas9 enzyme and sgRNA are constitutive promoters, and a second sgRNA is introduced into the same transformation cassette, but under the control of an inducible promoter. This second sgRNA can be designed to induce site-specific cleavage in the Cas9 gene to produce non-functional Cas9. In a more detailed embodiment, the second sgRNA induces cleavage at both ends of the transformation cassette, resulting in removal of the cassette from the host genome. This system provides controlled cell exposure time to the Cas enzyme and also minimizes off-target editing. Furthermore, it is possible to create transgenes free _{T 0} plants having a biallelic mutations using cutting across the CRISPR / Cas cassette (e.g., Moore et al, Nucleic Acids Research , 2014;. Schaeffer et al. , Plant Science, 2015). Moore et al. Method can be applied to the CRISPR / Cas9 system described herein.

Improved Plants The present invention also provides plants and yeast cells that can and are obtained by the methods provided herein. The improved plants obtained by the methods described herein are used in food or feed production, for example, by expressing genes that ensure resistance to plant pests, herbicides, dryness, low or high temperatures, excess water, etc. Can be useful.

Improved plants, especially crops and algae, obtained by the methods described herein are foods or feeds, for example by expression of higher protein, carbohydrate, nutrient or vitamin levels than would normally be found in the wild type. Can be useful in manufacturing. In this respect, improved plants, especially legumes and tubers, are preferred.

Other plants such as modified algae or oilseed rape may be particularly useful in the production of vegetable oils or biofuels, such as alcohols (particularly methanol and ethanol). They can be engineered to express or overexpress high levels of oils or alcohols used in the oil or biofuel industry.

The present invention also provides an improved portion of the plant. Plant sites include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperms, ovules, and pollen. The plant sites as envisioned herein may be viable, non-growth, reproducible, and / or non-renewable.

Also included herein is to provide plant cells and plants produced by the methods of the invention. Gametes, seeds, embryos (whether mating or somatic embryos), offspring or hybrids of plants containing genetic modifications produced by conventional breeding methods are also included within the scope of the invention. Such plants may contain heterologous or exogenous DNA sequences inserted into or in place of the target sequence. Alternatively, such plants may contain only certain changes (mutations, deletions, insertions, substitutions) in one or more nucleotides. As such, such plants differ only in the presence of certain modifications from their precursors.

Livestock and Producing Animals Therefore, the present invention provides plants, animals or cells produced by this method, or progeny thereof. The offspring may be clones of the produced plant or animal, or may result from sexual reproduction by mating with other individuals of the same species to introgress the desired trait into the offspring. The cells may be in vivo or ex vivo in the case of multicellular organisms, especially animals or plants.

Organisms and Animals; Methods The present application may also relate to other agricultural applications, such as livestock and production animals. Pigs, for example, have many features that make it attractive as a biomedical model, especially in regenerative medicine. In particular, pigs with severe combined immunodeficiency disease (SCID) can provide useful models for regenerative medicine, xenotransplantation, and tumorigenesis, as well as assist in the development of therapeutic agents for human SCID patients. Lee et al. , (Proc Natl Acad Sci US A. 2014 May 20; 111 (20): 7260-5) utilize a reporter-guided transcriptional activator-like effector nuclease (TALEN) system to affect both alleles. Targeted modifications of the recombinant activation gene (RAG) 2 were created in somatic cells with high efficiency, including those that exert. CRISPR Cas can be applied to similar systems.

Lee et al. , (Proc Natl Acad Sci US A. 2014 May 20; 111 (20): 7260-5) can be applied to the present invention as follows. Mutant pigs are generated by targeted modification of RAG2 in fetal fibroblasts followed by SCNT and embryo transfer. Fetal-derived fibroblasts are electroporated with constructs encoding CRISPR Cas and reporters. After 48 hours, transfected cells expressing green fluorescent protein are stored in individual wells of a 96-well plate as single cells with an estimated dilution per well. Targeted modifications of RAG2 are screened by amplifying genomic DNA fragments flanking any CRISPR Cas cleavage site and subsequently sequencing PCR products. After screening and confirming the absence of off-site mutations, cells carrying a targeted modification of RAG2 are used for SCNT. The polar body is removed with a portion of the adjacent cytoplasm of the oocyte (probably containing the metaphase metaphase plate of the second division), and the donor cell is placed around the yolk. The reconstituted embryos are then electroporated to fuse the donor cells with the oocytes and then chemically activate them. Activated embryos are incubated in 0.5 μM scriptide (S7817; Sigma-Aldrich) -containing porcine zygote medium 3 (PZM3) for 14-16 hours. The embryos are then washed to remove the scriptide and cultured in PZM3 until the embryos are transferred to the oviduct of the surrogate sow.

The present invention is also applicable to the modification of SNPs of other animals such as cows. Tan et al. (Proc Natl Acad Sci US A. 2013 Oct 8; 110 (41): 16526-16531) is a transcriptional activator-like (TAL) effector nuclease (TALEN) stimulant using plasmids, rAAV, and oligonucleotide templates. The livestock gene editing toolbox was developed to include clustered, regularly spaced short plasmid repeats (CRISPR) / Cas9-stimulated homology-dependent repair (HDR). Gene-specific gRNA sequences were cloned into the Church lab gRNA vector (Addgene ID: 41824) by their method (Mali P, et al. (2013), RNA-Guided Human Genome Engineering via Cas9 (RNA-Guided Human Genome Engineering via). Cas9) ". Science 339 (6121): 823-826). Cas9 nuclease was provided by cotransfection with either the hCas9 plasmid (Addgene ID: 41815) or mRNA synthesized from RSIScript-hCas9. This RSIScript-hCas9 was constructed by subcloning the XbaI-AgeI fragment from the hCas9 plasmid (including the hCas9 cDNA) into the RSIScript plasmid.

Heo et al. (Stem Cells Dev. 2015 Feb 1; 24 (3): 393-402. Doi: 10.1089 / scd. 2014.0278. Epub 2014 Nov 3) are bovine pluripotent cells and clustered regular intervals. We have reported highly efficient gene targeting in the bovine genome using short parindrome repeats (CRISPR) / Cas9 nucleases. First, Heo et al. Created induced pluripotent stem cells (iPSC) from bovine somatic fibroblasts by ectopic expression of Yamanaka factor and treatment with GSK3β and MEK inhibitor (2i). Heo et al. Observed that these bovine iPSCs are very similar to naive pluripotent stem cells in terms of gene expression and teratoma development potential. In addition, CRISPR / Cas9 nuclease specific for the bovine NANOG locus showed highly efficient editing of bovine genomes in bovine iPSCs and embryos.

Animality® is a cow for carrying out and transmitting traits of economic traits of economic importance such as carcass composition, carcass quality, maternal and reproductive traits and average daily weight gain. Provides profile analysis of animals such as. Analysis of the exhaustive Agency® profile begins with the discovery of DNA markers (most often single nucleotide polymorphisms or SNPs). All markers behind the Intelligence® profile have been discovered by independent scientists at universities, research organizations, and research institutions, including government agencies such as the USDA. The markers are then analyzed with Igency® in the validated population. Elementity® uses multiple resource populations representing different production environments and biological types to collect phenotypes that are not generally available in the beef industry seedstock, cow-calf, feed. Often collaborate with industrial partners from lots and / or packing segments. The bovine genome database is widely available, see, for example, the NAGRP Bovine Genome Coordination Program (http://www.animalgenome.org/cattle/maps/db.html). Therefore, the present invention can be applied to the targeting of bovine SNPs. Those skilled in the art, for example, Tan et al. Or Heo et al. As described above, the above protocols can be utilized for SNP targeting and they can be applied to bovine SNPs.

Qingjian Zou et al. (Journal of Molecular Cell Biology Advance October Public October 12, 2015) increases muscle mass in dogs by targeting the first exon of the inumiostatin (MSTN) gene (a negative regulator of skeletal muscle mass). Demonstrated. First, the efficiency of sgRNA was verified using cotransfection of canine embryonic fibroblasts (CEFs) with an sgRNA targeting MSTN with a Cas9 vector. MSTN KO dogs were then created by microinjecting a mixture of Cas9 mRNA and MSTN sgRNA into normal morphological embryos and autotransplanting zygotes into the oviducts of the same female dog. Knockout puppies showed a clear muscular phenotype on the femur compared to their wild-type littermates.

Livestock-Pigs Viral targets in livestock may, in some embodiments, include porcine CD163 on porcine macrophages, for example. CD163 is associated with infection by PRRSv (arterivirus porcine reproductive and respiratory disorder syndrome virus) (believed to be mediated by the virus's invasion into cells). Previously incurable porcine syndrome ("Mystery porcine disease"), when PRRSv is particularly infected with porcine alveolar macrophages (found in the lungs), causes damage, including reproductive disorders, weight loss and high mortality in domestic pigs Or "blue ear disease") is caused. Opportunistic infections such as epidemic pneumonia, meningitis and ear edema are often found due to immunodeficiency due to loss of macrophage activity. It also has significant economic and environmental impacts (estimated $ 660 million annually) due to increased use of antibiotics and financial losses.

In collaboration with Genus PLC, Christin M Whiteworth and Dr Randall Prather et al. Of the University of Missouri. CD163 was targeted with CRISPR-Cas9 and the edited porcine offspring were resistant to exposure to PRRSv, as reported by (Nature Biotechnology 3434 Online Publication 07 December 2015). Offspring were produced by breeding one founder male and one founder female (both having a mutation in exon 7 of CD163). The founder male had a 11 bp deletion in exon 7 on one allele, resulting in a frameshift mutation and a missense translation at amino acid 45 in domain 5 followed by an immature stop codon following amino acid 64. The other allele had an addition of 2 bp to exon 7 and a deletion of 377 bp to the preceding intron, which is expected to result in the expression of the first 49 amino acids of domain 5 followed by the immature termination code of amino acid 85. Was done. The sow had an addition of 7 bp to one of the alleles, which was expected to express the first 48 amino acids of domain 5 upon translation, followed by the immature stop codon of amino acid 70. The other allele of the sow was unamplifiable. The selected offspring were expected to be null animals (CD163 / −), ie CD163 knockouts.

Thus, in some embodiments, porcine alveolar macrophages can be targeted by the CRISPR protein. In some embodiments, porcine CD163 can be targeted by the CRISPR protein. In some embodiments, the porcine CD163 is by induction of DSB or by insertion or deletion, including one or more of those described above, eg, targeting a deletion or modification of exon 7. , Or can be knocked out by deletion or modification of exon 5 in other regions of the gene.

Edited pigs and their progeny, such as CD163 knockout pigs, are also envisioned. This can be livestock, breeding or modeling purposes (ie pig model). Semen containing gene knockout is also provided.

CD163 is a member of the scavenger receptor cysteine rich (SRCR) superfamily. Based on in vitro studies, SRCR domain 5 of this protein is the domain involved in the unpackaging and release of the viral genome. Therefore, other members of the SRCR superfamily can also be targeted to assess resistance to other viruses. PRRSV is also a member of the mammalian arterivirus group, which also includes mouse lactate dehydrogenase-elevating virus, deltaarterivirus vasovirus, and equine arteritis virus. Arteriviridae share important etiological characteristics, including the ability to cause both macrophage tropism and severe illness and persistent infection. Thus, arterivirus, and more specifically mouse lactate dehydrogenase-elevating virus, deltaarterivirus vasovirus and horse arteritis virus, have been targeted via, for example, porcine CD163 or other species and their homologues in mouse, monkey and horse models. Well, and knockouts are also provided.

In fact, this technique involves other livestock that can be transmitted to humans, such as swine flu virus (SIV) strains, which include swine flu C and subtypes of swine flu known as H1N1, H1N2, H2N1, H3N1, H3N2, and H2N3. It can extend to the disease and the viruses or bacteria that cause the above-mentioned pneumonia, meningitis and edema.

Xenografts, Xenografts The CRISPR-Cas system described herein, also for providing RNA-guided DNA nucleases adapted for use in providing modified tissue for transplantation. For example, the use of Cas9 effector protein system is also intended. For example, by using RNA-guided DNA nucleases to disrupt the expression of genes encoding epitopes recognized by the human immune system, ie heterologous antigen genes, transgenic pigs and the like (human hemoxygenase-1 transgenic). Can knock out, knock down or disrupt genes of choice in animals (such as porcine strains). Candidate porcine genes for disruption include, for example, α (1,3) -galactosyltransferase and cytidine monophosphate-N-acetylneuraminate hydroxylase genes (see International Publication No. 2014/066505). it can. In addition, genes encoding endogenous retroviruses, such as genes encoding all porcine endogenous retroviruses, may be disrupted (Yang et al., 2015, "Genome Wide of Pig Endogenous Retrovirus (PERV)). Gene-wise inactivation of porcine endogenus retroviruses (PERVs), Science 27 November 2015: Vol. 350 no. 6264 pp. 1101-1104). In addition, RNA-guided DNA nucleases can be used to improve protection from hyperacute rejection by targeting additional gene integration sites in xenograft donor animals, such as the human CD55 gene.

Gene Drives and Applications to Mosquitoes and Malaria Also described herein to provide RNA-guided gene drives in systems similar to those described in, for example, WO 2015/105928. We also contemplate the use of CRISPR-Cas systems such as Cas9 effector protein systems. This type of system can provide a method of altering eukaryotic germline cells, for example by introducing RNA-guided DNA nucleases and nucleic acid sequences encoding one or more guide RNAs into the germline cells. Guide RNAs can be designed to be complementary to one or more target locations on the genomic DNA of germline cells. The RNA-guided DNA nuclease-encoding nucleic acid sequence and the guide RNA-encoding nucleic acid sequence were arranged between flanking sequences on the construct so that germline cells could express the RNA-guided DNA nuclease and the guide RNA. It may be provided with a promoter and with any desired cargo coding sequence that is also located between the flanking sequences. The flanking sequence can typically contain the same sequence as the corresponding sequence on the target chromosome of selection, so that the flanking sequence works with the components encoded by the construct and by mechanisms such as homologous recombination. The insertion of the exogenous nucleic acid construct sequence into the genomic DNA at the target cleavage site is promoted and the germline cells homologate with respect to the exogenous nucleic acid sequence. In this way, the gene drive system has the ability to transfer the desired cargo gene throughout the breeding population (Gantz et al., 2015, "Very efficient Cas9 for population modification of the malaria vector mosquito stefence hamadaraka". mediated gene drive (Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi) ", PNAS 2015, published ahead of print November 23,2015, doi: 10.1073 / pnas.1521077112; Esvelt et al. , 2014, "Concerning RNA-guided gene drives for the vectoration of wild populations" eLife 2014; 3: e03401). In the embodiment of selection, a target sequence with few potential off-target sites in the genome can be selected. Targeting multiple sites within a target locus with multiple guide RNAs can increase the frequency of cleavage and prevent the evolution of drive-resistant alleles. Off-target cleavage can be reduced with truncated guide RNAs. A pair of nickases may be used instead of a single nuclease to further increase specificity. The gene drive construct may include a cargo sequence encoding a transcriptional regulator, eg, to activate a homologous recombination gene and / or suppress non-homologous end binding. Non-homologous end-binding events can cause lethality rather than producing drive-resistant alleles, as the target site can be selected within the essential gene. Gene drive constructs can be engineered to function in a host range over a temperature range (Cho et al. 2013, "Rapid, Adjustable Control of Protein Stability in Small Molecules in C. elegans (Rapid). and Tunable Control of Protein Stability in Caenorhabditis elegans Elegans Using a Small Molecule) ”, PLos ONE 8 (8): e72393.doi: 10.1371 / journ.

Illustrative Use of FISH and Inactivated CRISPR Cas9 Enzymes In one aspect, the invention presents the catalytically inactivated Cas protein described herein, preferably inactivated Cas9 ( Provided are engineered non-naturally occurring CRISPR-Cas systems containing dCas9), and the use of this system in fluorescence in situ hybridization (FISH). By fusing dCas9, which is incapable of causing DNA double-strand breaks, with a fluorescent protein such as high-sensitivity green fluorescent protein (eEGFP) and co-expressing it with a small guide RNA, it is interstitial and centromere in vivo. And can target terrorist repeats. The dCas9 system can be used to visualize both repetitive sequences and individual genes in the human genome. Such a new application of the labeled dCas9 CRISPR-cas system may be important in cell imaging and study of functional nuclear structure, especially when involving small nuclear volume or complex three-dimensional structures (Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B. 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR / Cas system ”. Cell 155 (7): 1479-91.doi: 10.016 / j.cell.

Therapeutic targeting with RNA-guided effector protein complexes As is clear, it is envisioned that this system can be used to target polynucleotide sequences of any interest. The present invention is a non-naturally occurring or engineered composition used to modify a target cell in vivo, ex vivo or in vitro, or one or more polynucleotides encoding a component of said composition, or said. A vector or delivery system containing one or more polynucleotides encoding a component of the composition is provided, and after modification, the cell so that the progeny or cell line of the cell modified by CRISPR retains the altered phenotype. Can be done in a variable manner. The modified cells and progeny can be part of a multicellular organism such as a plant or animal in which the CRISPR system has been applied ex vivo or in vivo to the desired cell type. The CRISPR invention can be a therapeutic method. Therapeutic treatment methods may include gene or genome editing, or gene therapy.

Treatment of Pathogens such as Bacterial, Fungal and Parasitic Pathogens The present invention may also be applied to the treatment of bacterial, fungal and parasitic pathogens. Although most studies focus on the development of new antibiotics, new antibiotics are nevertheless exposed to the same drug resistance problems after development. The present invention provides a novel CRISPR-based alternative that solves these challenges. Moreover, unlike existing antibiotics, CRISPR-based therapies can be pathogen-specific and can induce bacterial cell death of the target pathogen while avoiding beneficial bacteria.

Jiang et al. (“RNA-guided editing of bacterial genomes CRISPR-Cas systems”, Nature Biotechnology vol.31, p.233-9, MarchCR20, MarchCR20, CRISPR-Cas system. -The Cas9 system was used to mutate or kill S. pneumoniae and E. coli. This study, which introduced accurate mutations into the genome, relies on killing non-mutated cells by dual RNA: Cas9-guided cleavage at the target genome site, and avoids the need for selectable markers or counterselective systems. It is a thing. Antibiotic resistance has been reversed using the CRISPR system, eliminating the transfer of resistance between strains. Biccard et al. Showed that Cas9, reprogrammed to target the virulence gene, kills toxic but non-toxic Staphylococcus aureus (S. aureus). When the nuclease was reprogrammed to target the antibiotic resistance gene, the staphylococcal plasmid containing the antibiotic resistance gene was disrupted and immune to the spread of the resistance gene carried by the plasmid (Bikard et al.,. "Producing Sequence-Specific Antibacterial Agents Using CRISPR-Cas nuclease (Exploiting CRISPR-Cas Nucleases to Products-Specific Antimicrobials)", Nature Biotechnology, Vol. 1,150,03,304. Published online 05 October 2014). Bikard has shown that CRISPR-Cas9 antibiotics function to kill Staphylococcus aureus (S. aureus) in vivo in a mouse skin colony forming model. Similarly, Yosef et al. Used the CRISPR system to target genes encoding enzymes that confer resistance to β-lactam antibiotics (Yousef et al., “Inspiring antibiotic-resistant bacteria”. Temperate and lytic bacteriophages programmed to sense and kill antibiotic-resistant bacteria, Proc. Natl. 72, Proc. Nat. 72. doi: 10.1073 / pnas. 1500107112 Online publication May 18, 2015).

The CRISPR system can be used to edit the genome of parasites that are resistant to other genetic techniques. For example, the CRISPR-Cas9 system has been shown to introduce double-strand breaks into the Plasmodium yoelii genome (Zhang et al., "Efficiency of the Plasmodium parasite genome using the CRISPR / Cas9 system." (See Effective Editing of Malaria Parasite Genome Using the CRISPR / Cas9 System), mBio.vol.5, e01414-14, Jul-Aug 2014). Ghorbal et al. ("Genome editing in the human malaria parasite Plasmodium falciparumusing the CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system. 821, doi: 10.1038 / nbt.2925, published online June 1,2014) sequences of two genes, orc1 and kelch13, which have putative roles in the development of resistance to gene silence and artemicinin, respectively. Was modified. This system can be used to generate neutral or even adverse mutations, as parasites altered at appropriate sites recovered with extremely high efficiency, even though there was no direct choice for modification. Is suggested. CRISPR-Cas9 has also been used to modify the genome of other pathogenic parasites, including Toxoplasma gondii (Shen et al., "Efficient gene disruption in various Toxoplasma gondii strains". Disruption in diversity of Toxoplasma gondii using CRISPR / CAS9) ”, mBio vol.5: e01114-14,2014; and Sideik et al.,“ CRISPR / Cas9-based Toxoplasma gondii Genome Efficient Toxoplasma gondii of Toxoplasma gondii Using CRISPR / Cas9) ”, PLoS One vol.9, e100450, doi: 10.1371 / genome.pone.0104505, published online June 27, 2014).

Vyas et al. ("A Candida albicans CRISPR system enables genetic engineering of essential genes and gene families (A Candida alvicans CRISPR system permits genetic engineering of essential genes and genes1, genes, genes, genes, genes, genes, genes, genes, genes. 1126 / sciadv. 1500248, April 3, 2015), using the CRISPR system, C.I. It eliminates many years of genetic engineering obstacles in C. albicans and efficiently mutates both copies of several different genes in a single experiment. In organisms in which several mechanisms contribute to drug resistance, Vyas generated homozygous double mutants that no longer exhibit the super-resistance to fluconazole or cycloheximide exhibited by the parent clinical isolate Can90. Vyas also said C.I. Homozygous loss-of-function mutations were also obtained by creating conditional alleles in the essential genes of C. albicans. The DCR1 null allele required for ribosomal RNA processing is lethal at low temperatures but survives at high temperatures. Vyas used a repair template to introduce a nonsense mutation and isolated a dcr1 / dcr1 mutant that could not grow at 16 ° C.

The CRISPR system of the present invention used for Plasmodium falciparum (P. falciparum) by disrupting chromosomal loci. Ghorbal et al. ("Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system, CRISPR-Cas9 system ), DOI: 10.1038 / nbt.2925, June 1,2014) introduced gene knockouts and single nucleotide substitutions specific for the Malaria genome using the CRISPR system. To adapt the CRISPR-Cas9 system to Plasmodium falciparum (P. falcipalum), Ghorbal et al. Plasmodium falciparum is a plasmid regulatory element of malaria protozoa in the pUF1-Cas9 episome, which also has the drug-selectable marker ydhodh that confer resistance to the P. falcipalum dihydroorotoic acid dehydrogenase (PfDHODH) inhibitor DSM1. An expression vector was prepared to be controlled, and for transcription of sgRNA, a guide RNA and a donor DNA template for homologous recombination repair were placed on the same plasmid, pL7, and Plasmodium falciparum U6 A small nuclear RNA (sn) RNA regulatory element was used. In addition, Zhang C.I. et al. ("Efficient editing of malaria parasite genome using the CRISPR / Cas9 system", MBio, 2014 Jul 1: 5 (4): E014- 10.1128 / MbIO.01414-14) and Wagner et al. ("Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum", Nature Methods: 11,915-918 (2014), 915-918 (2014), DO See also .3063).

Treatment of Pathogens such as Viral Pathogens such as HIV Cas-mediated genome editing can be used to introduce protective mutations into somatic cell tissues to combat non-genetic or complex diseases. For example, NHEJ-mediated inactivation of CCR5 receptors in lymphocytes (Lombardo et al., Nat Biotechnol. 2007 Nov; 25 (11): 1298-306) is a viable strategy for avoiding HIV infection. On the other hand, PCSK9 (Cohen et al., Nat Genet. 2005 Feb; 37 (2): 161-5) or angiopoetin (Musunuru et al., N Engl J Med. 2010 Dec 2; 363 (23): 2220- Deletion of 7) can provide a therapeutic effect on statin-resistant hypercholesterolemia or hyperlipidemia. These targets can also be addressed with siRNA-mediated protein knockdown, but the unique advantage of NHEJ-mediated gene inactivation is the ability to achieve permanent therapeutic benefits without the need to continue treatment. Is. As with all gene therapies, of course, it can be important to establish that each proposed therapeutic use has a favorable risk-benefit ratio.

When plasmid DNA encoding Cas9 and guide RNA was hydrodynamically delivered to the liver of an adult mouse tyrosinemia model together with a repair template, mutation Fah gene modification in about 1 in 250 cells and wild-type Fah protein It has been shown that rescue of expression is possible (Nat Biotechnol. 2014 Jun; 32 (6): 551-3). In addition, in clinical trials, ZF nuclease was successfully used to combat HIV infection by exovibon knockout of the CCR5 receptor. HIV DNA levels decreased in all patients and HIV RNA became undetectable in 1 in 4 patients (Thebes et al., N Engl J Med. 2014 Mar 6; 370 (10): 901-10. ). Both of these results demonstrate the promise of programmable nucleases as a novel therapeutic platform.

In another embodiment, a self-inactivating lentiviral vector comprising a siRNA targeting a common exon shared by HIV tat / rev, a nucleolus localized TAR decoy, and an anti-CCR5-specific hammerhead ribozyme (eg,). DiGiusto et al. (2010) Sci Transfer Med 2: 36 ra43) may be used and / or adapted to the CCRISP-Cas9 system of the invention. Collect at least 2.5 × 10 ⁶ CD34 + cells per kilogram of patient body weight, 2 μmol / L-glutamine, stem cell factor (100 ng / ml), Flt-3 ligand (Flt-3L) (100 ng / ml) , And thrombopoietin (10 ng / ml) (CellGenix) can be pre-stimulated in X-VIVO 15 medium (Lonza) at a density of 2 × 10 ⁶ cells / ml for 16-20 hours. Pre-stimulated cells can be transduced with lentivirus at a multiplicity of infection of 5 for 16-24 hours in a 75 cm ² tissue culture flask coated with fibronectin (25 mg / cm ² ) (RetroNectin, Takara Bio Inc.).

Using knowledge in the art and the teachings of the present disclosure, one of ordinary skill in the art can modify HSCs for immunodeficiency conditions such as HIV / AIDS, and put HSCs into the CRISPR-Cas9 system, which targets and knocks out CCR5. Includes contacting steps. Guide RNAs that target and knock out particles containing CCR5- and -Cas9 proteins (and advantageously dual guide techniques, eg different guide RNA pairs; eg, primary human CD4 + T cells and CD34 + hematopoietic stems and precursor cells (HSPC). ), Two clinically related genes, a guide RNA that targets B2M and CCR5), are contacted with HSC. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier. In addition, Kiem, "Hematopoietic stem cell-based gene therapy for HIV disease", Cell Stem Cell. Feb 3, 2012; 10 (2): 137-147 (incorporated herein by reference with the references cited therein); Mandal et al, "Genes in human hematopoietic stem and effector cells with CRISPR / Cas9. Efficient Ablation (Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR / Cas9) ”, Cell Stem Cell, Volume 15, Issue 5, p643-652, Also referred to herein). In addition, as another means of countering HIV / AIDS using the CRISPR-Cas9 system, Ebina, "CRISPR / Cas9 system (CRISPR / Cas9) that suppresses HIV-1 expression by editing the HIV-1 integrated provirus DNA. CRISPR to support HIV-1 expression by editing HIV-1 integrated proviral DNA) ”SCIENTIFIC REPORTS | 3:2510 | DOI: 10.1038 / rep02510 (also referred to herein by reference). Be done.

The rationale for genome editing for the treatment of HIV is that individuals homozygous for loss-of-function mutations in CCR5, a cell co-receptor to the virus, are extremely resistant to infection and are otherwise healthy and genomic. It derives from observations that editing suggests that mimicking this mutation may be a safe and effective therapeutic strategy [Liu, R. et al. , Et al. Cell 86, 367-377 (1996)]. The idea was that when HIV-infected patients received an allogeneic bone marrow transplant from a homozygous donor for a loss-of-function CCR5 mutation, undetectable levels of HIV and normal CD4 T-cell count recovery were achieved. The clinical validity was confirmed by [Hutter, G. et al. , Et al. The New England journal of medicine 360, 692-698 (2009)]. Bone marrow transplantation is not a viable therapeutic strategy for many HIV patients due to its high cost and potential graft-versus-host disease, but HIV treatments that convert patients' own T cells to CCR5 are desirable.

Early studies of knocking out CCR5 with ZFNs and NHEJs in humanized mouse HIV models have shown that transplantation of CCR5-edited CD4 T cells improves viral load and CD4 T cell count [Perez, E. et al. E. , Et al. Nature biotechnology 26,808-816 (2008)]. Importantly, these models also showed that HIV infection provided a choice for CCR5 null cells, giving fitness superiority by editing and potentially a small number of editing cells therapeutically effective. It is suggested that it will be possible to produce.

As a result of this and other promising preclinical studies, genome editing therapies that knock out CCR5 in patient T cells are currently being tested in humans [Holt, N. et al. , Et al. Nature biotechnology 28,839-847 (2010); Li, L. et al. , Et al. Molecular therapy: the journal of the American Society of Gene Therapy 21, 1259-1269 (2013)]. In a recent Phase I clinical trial, CD4 + T cells were removed from HIV patients, edited with ZFNs designed to knock out the CCR5 gene, and returned to patients by autologous transplantation [Tebas, P. et al. , Et al. The New England journal of medicine 370, 901-910 (2014)].

In another study (Mandal et al., Cell Stem Cell, Volume 15, Issue 5, p643-652, 6 November 2014), two clinically associated in human CD4 + T cells and CD34 + hematopoietic stem and progenitor cells (HSPC). Sexual genes, B2M and CCR5, are targeted by CRISPR-Cas9. Using a single RNA guide, HSPC gave extremely high efficiency mutagenesis, but not T cells. The dual-guided approach improved the effectiveness of gene deletions in both cell types. HSPC in which genome editing by CRISPR-Cas9 occurred retained pluripotency. When the expected on-target and off-target mutations in HSPC were examined by target capture sequencing, low levels of off-target mutagenesis were observed in only one site. These results demonstrate that CRISPR-Cas9 can efficiently ablate genes in HSPC while minimizing off-target mutagenesis, which has wide applicability to hematopoietic cell-based therapies. is there.

Wang et al. (PLoS One. 2014 Dec 26; 9 (12): e115987. Doi: 10.1371 / journal. Pone. 0115987), Cas9 and CCR5 by CRISPR-related protein 9 (Cas9) and single-guided RNA (guide RNA). CCR5 was silenced with a lentiviral vector expressing guide RNA. Wang et al. Showed that one round of transduction of a lentiviral vector expressing Cas9 and CCR5 guide RNA into HIV-1 sensitive human CD4 + cells resulted in frequent CCR5 gene disruption. Cells in which the CCR5 gene has been disrupted are not only resistant to R5-trophic HIV-1, including transmission / founder (T / F) HIV-1 isolates, but also have the CCR5 gene upon R5-trophic HIV-1 infection It also has a selective advantage over cells that have not been destroyed. In stably transduced cells, genomic mutations at potential off-target sites showing a high degree of homology with these CCR5 guide RNAs were detected in the T7 endonuclease I assay, even 84 days after transduction. There wasn't.

Fine et al. (Sci Rep. 2015 Jul 1; 5: 10777. Doi: 10.1038 / rep10777) is a Streptococcus pyogenes (S. A two-cassette system expressing a pyogenes) Cas9 (SpCas9) protein fragment was identified. Fine et al. Demonstrated the effectiveness of this system as a single Cas9 and as a Cas9 nickase pair in cleavage of the HBB and CCR5 genes in human HEK293T cells using a specific CRISPR guide strand. Trans-spliced SpCas9 (tsSpCas9) showed approximately 35% nuclease activity compared to wild-type SpCas9 (wtSpCas9) at standard transfection doses, but was substantially more active at lower dose levels. It has decreased. Since tsSpCas9 has a significantly reduced open reading frame length compared to wtSpCas9, it potentially leads to AAV vectors containing tissue-specific promoters, multiplexed guide RNA expression, and effector domain fusions with SpCas9. It is possible to package more complex and longer genetic factors.

Li et al. (J Gen Virol. 2015 Aug; 96 (8): 2381-93. Doi: 10.1099 / vir. 0.000139. Epub 2015 Apr 8), CRISPR-Cas9 efficiently edits the CCR5 locus in cell lines. It has been demonstrated that it can be mediated and can result in knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the expected cleavage site of CCR5. No significant off-target effect was detected at the 15 top-scoring potential sites for each of the three most effective guide RNAs analyzed. Li et al. By constructing a chimeric Ad5F35 adenovirus having a CRISPR-Cas9 component, it efficiently transduces primary CD4 + T lymphocytes and disrupts CCR5 expression, and HIV-1 resistance to transduced-positive cells. Gender was given.

WO 2015/148670 Pamphlet is mentioned, and through the teachings of this specification, the present invention includes the methods and materials of this document that apply in conjunction with the teachings of this specification. In certain aspects of gene therapy, methods and compositions for editing target sequences associated with or associated with the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) are contemplated. In a related aspect, the inventions described herein include the prevention and treatment of HIV infection and AIDS by introducing one or more mutations into the gene for CC chemokine receptor type 5 (CCR5). To do. The CCR5 gene is also known as CKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22, and CC-CKR-5. In a further aspect, the inventions described herein provide prevention or reduction of HIV infection and / or prevention or reduction of the ability of HIV to invade host cells, eg, in a subject already infected. Include. Illustrative host cells for HIV include, but are not limited to, CD4 cells, T cells, gut-associated lymphoid tissue (GALT), macrophages, dendritic cells, bone marrow progenitor cells, and microglia. Viral invasion into host cells requires interaction of the viral glycoproteins gp41 and gp120 with both CD4 and co-receptors, such as CCR5. If a co-receptor, such as CCR5, is not present on the surface of the host cell, the virus cannot bind to and invade the host cell. Therefore, the progression of the disease is hindered. Invasion of the HIV virus into the host cell is prevented by knocking out the CCR5 of the host cell or by knocking down, for example, by introducing a protective mutation (such as the CCR5 delta 32 mutation).

Those skilled in the art can describe the targeting of CCR5 by the CRISPR Cas9 system of the present invention, eg, Holt, N. et al. , Et al. Nature biotechnology 28,839-847 (2010), Li, L. et al. , Et al. Molecular therapy: the journal of the American Society of Gene Therapy 21, 1259-1269 (2013), Mandal et al. , Cell Stem Cell, Volume 15, Issue 5, p643-652, 6 November 2014, Wang et al. (PLoS One. 2014 Dec 26; 9 (12): e115987. Doi: 10.1371 / journal. Pone. 0115987), Fine et al. (Sci Rep. 2015 Jul 1; 5: 10777. Doi: 10.1038 / srep 10777) and Li et al. The above study of (J Gen Virol. 2015 Aug; 96 (8): 2381-93. Doi: 10.1099 / vir. 0.000139. Epub 2015 Apr 8) can be utilized.

Treatment of pathogens such as viral pathogens such as HBV Chronic hepatitis B virus (HBV) infection is widespread, fatal, and rarely cured due to persistent viral episomal DNA (cc cDNA) in infected cells. Ramana et al. (Ramanan V, Shromai A, Cox DB, Schwartz RE, Michiaridis E, Bhatta A, Scott DA, Zhang F, Rice CM, Bhatia SN, Scientific Rep.2015 Jun 2; 5: 10833. Online publication 2nd June 2015.) showed that the CRISPR / Cas9 system can specifically target and cleave conserved regions of the HBV genome, resulting in robust suppression of viral gene expression and replication. The inventors have noted that Cas9 cleaves ccDNA during sustained expression of Cas9 and properly selected guide RNAs and that both ccDNA and other parameters of viral gene expression and replication are dramatically reduced. Indicated. Therefore, the inventors have shown that direct targeting of viral episomal DNA is a novel therapeutic technique that controls the virus and potentially cures the patient. It is also described in Broad Institute et al. It is also described in Pamphlet International Publication No. 2015089465 A1 in the name of (this content is incorporated by reference herein).

The present invention can also be applied to the treatment of hepatitis B virus (HBV). However, the CRISPR Cas system must be adapted, eg, by optimizing the dose and sequence, to avoid RNAi drawbacks such as the risk of the endogenous small RNA pathway becoming oversaturating (eg,). , Grimm et al., Nature vol. 441, 26 May 2006). For example, low doses such as about 1-10 × 10 ¹⁴ particles per human are contemplated. In another embodiment, a CRISPR Cas system targeting HBV can be administered in liposomes such as stable nucleic acid lipid particles (SNALP) (eg, Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August). See 2005). Approximately 1, 3 or 5 mg / kg / day in SNALP, HBV RNA-targeted CRISPR Cas is contemplated for daily intravenous injection. Daily treatment is obtained for about 3 days, then weekly, for about 5 weeks. In another embodiment, Chen et al. System (Gene Therapy (2007) 14, 11-19) can be used and / or adapted to the CRISPR Cas system of the present invention. Chen et al. Delivers shRNA using a double-stranded adeno-associated virus type 8 pseudovector (dsAAV2 / 8). A single dose of dsAAV2 / 8 vector carrying HBV-specific shRNA (1 × 10 ¹² vector genome per mouse) effectively suppressed stable levels of HBV protein, mRNA and replication DNA in the liver of HBV transgenic mice. A maximum of 2-3 log ₁₀ reductions in circulating HBV load was achieved. Significant HBV suppression persisted for at least 120 days after vector administration. The therapeutic effect of shRNA was sequence-dependent and was not accompanied by interferon activation. In the present invention, the HBV-oriented CRISPR Cas system is cloned into an AAV vector such as the AAV2 / 8 vector and administered to humans at a dosage of, for example, about 1 × 10 ¹⁵ vector genome to about 1 × 10 ¹⁶ vector genome per human. Can be done. In another embodiment, Wooddell et al. (Molecular Therapy vol. 21 no. 5, 973-985 May 2013) can be used and / or adapted to the CRISPR Cas system of the present invention. Woodell et al. Is a simple combination of a hepatocyte-targeted N-acetylgalactosamine conjugated melitine-like peptide (NAG-MLP) and a hepatotrophic cholesterol-conjugated siRNA (chol-siRNA) that targets coagulation factor VII (F7). It is shown that infusion results in efficient F7 knockdown in mice and non-human primates without changes in clinical chemistry or induction of cytokines. Wooddell et al. Using a transient transgenic mouse model of HBV infection, a single co-injection of NAG-MLP with a potent chol-siRNA that targets conserved HBV sequences is a viral RNA, protein. , And the suppression of multilog of viral DNA, indicating that the effect lasted for a long time. In the present invention, for example, intravenous co-injection of about 6 mg / kg of NAG-MLP and 6 mg / kg of HBV-specific CRISPR Cas can be envisioned. In an alternative example, about 3 mg / kg of NAG-MLP and 3 mg / kg of HBV-specific CRISPR Cas were delivered on day 1, followed by about 2-3 mg / kg of NAG-MLP and 2-3 mg / kg two weeks later. kg of HBV-specific CRISPR Cas may be administered.

Lin et al. (Mol Ther Nucleic Acids. 2014 Aug 19; 3: e186. Doi: 10.1038 / mtna. 2014.38) designed eight gRNAs for HBV of genotype A. With HBV-specific gRNA, the CRISPR-Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with the HBV expression vector. Of the eight screened gRNAs, two valid ones were identified. A single gRNA targeting the conserved HBV sequence acted on a variety of genotypes. Lin et al. Using a hydrodynamics-HBV sustained mouse model, the system can achieve reduced serum surface antigen levels by cleaving the intrahepatic HBV genome-containing plasmid and promoting its clearance in vivo. Further demonstrated. These data suggest that the CRISPR-Cas9 system can disrupt HBV expression templates both in vitro and in vivo, demonstrating its potential in the eradication of persistent HBV infection.

Dong et al. (Antiviral Res. 2015 Jun; 118: 110-7. Doi: 10.016 / j. Antiviral. 2015.03.015.Epub 2015 Appr 3) targeted the HBV genome using the CRISPR-Cas9 system and HBV. Efficiently inhibited infection. Dong et al. Synthesized four single guide RNAs (guide RNAs) that target the conserved region of HBV. Expression of these guide RNAs with Cas9 reduced virus production in Huh7 cells and HBV replication cells HepG 2.2.15. Dong et al. Further demonstrated that CRISPR-Cas9 led to cleavage and cleavage-mediated mutagenesis occurred in the HBV ccDNA of transfected cells. In a mouse model with HBV ccDNA, rapid tail intravenous injection of the guide RNA-Cas9 plasmid reduced ccDNA and HBV protein levels.

Liu et al. (J Gen Virol. 2015 Aug; 96 (8): 2252-61. Doi: 10.1099 / vir. 0.000159. Epub 2015 Apr 22) targets eight conservative regions of various HBV genotypes. Guide RNAs (gRNAs) have been designed that can significantly inhibit HBV replication both in vitro and in vivo, thereby investigating the potential for disrupting HBV DNA templates using the CRISPR-Cas9 system. .. The HBV-specific gRNA / Cas9 system was able to inhibit the replication of HBVs of various genotypes in cells, and a single gRNA / Cas9 system significantly reduced viral DNA, resulting in various gRNA / Cas9 systems. Removed by combination.

Wang et al. (World J Gastroenterol. 2015 Aug 28; 21 (32): 9554-65. Doi: 10.3748 / wjg.v21.i 32.9554) designed 15 gRNAs for HBV of genotypes A to D. Eleven combinations (dual gRNAs) of the two above gRNAs covering the regulatory region of HBV were selected. By measuring the HBV surface antigen (HBsAg) or e-antigen (HBeAg) in the culture supernatant, the efficiency of each gRNA and 11 dual gRNAs for suppression of HBV (genotypes AD) replication was examined. Destruction of the HBV expression vector was examined using polymerase chain reaction (PCR) and sequencing methods in HuH7 cells cotransfected with dual gRNA and HBV expression vectors, and KCl precipitation, plasmid-safe ATP-dependent DNase in HepAD38 cells. Destruction of ccDNA was examined using a combination of (PSAD) digestion, rolling circle amplification and quantitative PCR. The cytotoxicity of these gRNAs was evaluated by the mitochondrial tetrazolium assay. All gRNAs were able to significantly reduce HBsAg or HBeAg production in the culture supernatant, which production was dependent on the region of interest for the gRNA. All dual gRNAs can efficiently suppress HBsAg and / or HBeAg production for HBVs of genotypes A to D, and the effectiveness of dual gRNAs in suppressing HBsAg and / or HBeAg production is the single gRNA alone. Significantly increased when compared to use. Furthermore, the Applicants confirmed that PCR direct sequencing was able to specifically disrupt the HBV expression template by removing fragments between the cleavage sites of the two gRNAs used by these dual gRNAs. did. Most importantly, the combination of gRNA-5 and gRNA-12 was able to not only efficiently suppress HBsAg and / or HBeAg production, but also disrupt the ccDNA reservoir in HepAD38 cells.

Karimova et al. (Sci Rep. 2015 Sep 3; 5: 13734. Doi: 10.1038 / rep13734) was a cross-genotype-conserved HBV targeted for specific and effective cleavage by Cas9 nickase in the S and X regions of the HBV genome. The sequence was identified. This technique disrupted not only episomal cc cDNA and chromosomally integrated HBV target sites in reporter cell lines, but also HBV replication in chronically and de novo-infected hepatocellular carcinoma cell lines.

Those skilled in the art can describe the targeting of HBV by the CRISPR Cas system of the present invention, for example, by Lin et al. (Mol Ther Nucleic Acids. 2014 Aug 19; 3: e186. Doi: 10.1038 / mtna. 2014.38), Dong et al. (Antiviral Res. 2015 Jun; 118: 110-7. Doi: 10.016 / j. Antiviral. 2015.03.015.Epub 2015 Appr 3), Liu et al. (J Gen Virol. 2015 Aug; 96 (8): 2252-61. Doi: 10.1099 / vir. 0.000159. Epub 2015 Appr 22), Wang et al. (World J Gastroenterol. 2015 Aug 28; 21 (32): 9554-65. Doi: 10.3748 / wjg. V21.i 32.9554) and Karimova et al. The above study of (Sci Rep. 2015 Sep 3; 5: 13734. Doi: 10.1038 / srep 13734) is available.

Patient-Specific Screening Methods Nucleotides, such as CRISPR-Cas systems that target trinucleotide repeats, can be used to screen patients or patient samples for the presence of such repeats. This repeat can be the target of RNA of the CRISPR-Cas system, and if there is binding to this repeat by the CRISPR-Cas system, the binding can be detected and thereby indicate the presence of such repeat. .. Therefore, the CRISPR-Cas system can be used to screen patients or patient samples for the presence of repeats. The condition can then be addressed by administering one or more compounds suitable for the patient; or the CRISPR-Cas system can be administered to bind and cause insertion, deletion, or mutation of the condition. Can be alleviated.

Treatment of diseases by genetic or epigenetic aspects Gluckmann et al. International Publication No. 2015/048577, CRISPR-related methods and compositions (CRISPR-RELATED METHODS AND COMPOSITIONS); Glucksmann et al, International Publication No. 2015/070083, pamphlet "CRISPR-related methods and compositions with controlling gRNA (CRISPR-related methods and compositions with controlling gRNA) CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS) ”, which is in the published application of Editas Medicine that describes how to treat the disease therapeutically with gene therapy using the Cas9 system at the target locus. Gene mutations that have been attempted using TALEN and ZFN, including, but with limited success and have been identified as potential targets for the Cas9 system, using the CRISPR-Cas9 system of the invention. Can be repaired.

Maeder et al. International Publication No. 2015/134812 pamphlet "CRISPR / CAS-related methods and compositions for the treatment of Usher syndrome and retinitis pigmentosa (CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING USHER SYNDROME AND RETITISIS PIGMENT" Be done. Through the teachings of this specification, the present invention includes the methods and materials of these documents that apply in conjunction with the teachings of this specification. In certain embodiments of ocular and auditory gene therapy, methods and compositions for the treatment of Usher syndrome and retinitis-pigmentosa may be adapted to the CRISPR-Cas system of the invention (eg, international publication). See Pamphlet No. 2015/134812). In certain embodiments, WO 2015/134812 corrects a guanine deletion at position 2299 of the USH2A gene using gene editing, eg, a CRISPR-Cas9 mediated method (eg, lack of position 2299 of the USH2A gene). It involves delaying the treatment or onset or progression of Usher syndrome type IIA (USH2A, USH11A) and retinitis pigmentosa type 39 (RP39) by (replacement of lost guanine residues). In a related embodiment, it is cleaved with one or more nucleases, one or more nickases, or a combination thereof, eg, thereby causing a point mutation (eg, deletion of a single nucleotide, eg guanine). Mutations are targeted by inducing HDR containing the donor template to be modified. Changes or modifications of the mutant USH2A gene may be mediated by any mechanism. Illustrative mechanisms that may be associated with alterations (eg, modification) of the mutant HSH2A gene include, but are not limited to, non-homologous end binding, microhomology-mediated end binding (MMEJ), homology-dependent repair (eg, endogenous donor template). Mediation), SDSA (Synthesis Dependent Chain Annealing), Single Chain Annealing or Single Chain Invasion. In certain embodiments, the method used for the treatment of Usher syndrome and retinitis pigmentosa comprises the step of obtaining information on the mutations that the subject has, eg, by sequencing the appropriate portion of the USH2A gene. obtain.

In some embodiments, treatment, prevention or diagnosis of primary open-angle glaucoma (POAG) is provided. The target is preferably the MYOC gene. This is described in Pamphlet International Publication No. 2015/153780, the disclosure of which is incorporated by reference herein.

Also mentioned is Pamphlet International Publication No. 2015/138510, and through the teachings of this specification, the present invention (using the CRISPR-Cas9 system) provides treatment, onset or progression of label congenital amaurosis type 10 (LCA 10). Includes providing delaying. LCA 10 is a mutation in the CEP290 gene, eg, c.I. 2991 + 1655, caused by a mutation from adenine to guanine. This is a mutation in nucleotide 1655 of intron 26 of CEP290, eg, a mutation from A to G. CEP290 is also known as CT87; MKS4; POC3; rd16; BBS14; JBTS5; LCAJO; NPHP6; SLSN6; and 3H11Ag (see, eg, WO 2015/138510). In some aspects of gene therapy, the invention comprises introducing one or more cut points in the vicinity of the site of the LCA target location in at least one allele of the CEP290 gene (eg, c.2991 + 1655; A to G). ). Changes in the LCA10 target position include (1) cleavage-induced indel introduction (also referred to herein as NHEJ-mediated indel introduction) that is very close to or includes the LCA10 target position (eg, c.2991 + 1655A to G). ) Or (2) Cleavage-induced deletion of genomic sequences (also referred to herein as NHEJ-mediated deletions), including mutations at the LCA10 target location (eg, c.2991 + 1655A to G). .. Both methods result in deletion or destruction of the cryptosplice site due to mutations at the LCA 10 target location.

In some embodiments, the present invention (using the CRISPR-Cas9 system) includes providing treatment or delaying the onset or progression of Label Congenital Amaurosis Type 10 (LCA 10). LCA 10 is a mutation in the CEP290 gene, eg, c.I. 2991 + 1655, caused by a mutation from adenine to guanine. This is a mutation in nucleotide 1655 of intron 26 of CEP290, eg, a mutation from A to G. CEP290 is also known as CT87; MKS4; POC3; rd16; BBS14; JBTS5; LCAJO; NPHP6; SLSN6; and 3H11Ag (see, eg, WO 2015/138510). In some aspects of gene therapy, the invention comprises introducing one or more cut points in the vicinity of the site of the LCA target location in at least one allele of the CEP290 gene (eg, c.2991 + 1655; A to G). ). Changes in the LCA10 target position include (1) cleavage-induced indel introduction (also referred to herein as NHEJ-mediated indel introduction) that is very close to or includes the LCA10 target position (eg, c.2991 + 1655A to G). ) Or (2) Cleavage-induced deletion of genomic sequences (also referred to herein as NHEJ-mediated deletions), including mutations at the LCA10 target location (eg, c.2991 + 1655A to G). .. Both methods result in deletion or destruction of the cryptosplice site due to mutations at the LCA 10 target location.

Researchers are wondering if gene therapy can be used to treat a variety of diseases. The CRISPR system of the invention based on the Cas9 effector protein is envisioned for such therapeutic applications, including, but not limited to, further exemplified target ranges, and with delivery methods such as: Some examples of pathologies or diseases that could be usefully treated with this system are included in the examples of genes and references contained herein, and are currently associated with those pathologies, as well. Provided there. The exemplified genes and pathologies are not exhaustive.

Treatment of Cardiovascular Diseases The present invention also contemplates delivering the CRISPR-Cas9 system, specifically the novel CRISPR effector protein system described herein, to blood or hematopoietic stem cells. Wahlgren et al. Plasma exosomes (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) have been previously described and can be utilized to deliver the CRISPR Cas9 system to the blood. The nucleic acid targeting system of the present invention is also intended to treat abnormal hemoglobinosis, such as thalassemia and sickle cell disease. For example, see WO 2013/126794 for potential targets that can be targeted by the CRISPR Cas9 system of the invention.

Drakopoulou, "Review Paper, Ongoing Challenges of Hematopoietic Stem Cell-Based Gene Therapy for β-Saracemia (Review Article, The Ongoing Challenge of Hematopoietic Stem Cell-Based Gene Therapy 201, Gene Therapy) ID 987980, 10 pages, doi: 10.4061/2011/987980 (incorporated herein by reference as all indicated, together with its references) deliver the gene for β-globin or γ-globin. We are considering the modification of HSC using a lentivirus. In contrast to using lentivirus, those skilled in the art, based on their knowledge in the art and the teachings of the present disclosure, target and modify mutations in the β-thalassemia CRISPR-Cas9 system (eg β- HSCs can be modified using globin or γ-globin, preferably including a suitable HDR template that delivers the coding sequence for non-sickle sickle β-globin or γ-globin; , Guide RNA can target mutations that give rise to β-thalassemia, and HDR can result in coding of appropriate β-globin or γ-globin expression. The mutated HSC is contacted with a guide RNA that targets particles containing the mutated-and-Cas9 proteins. The particles may also contain an HDR template suitable for modifying the mutation for proper expression of β-globin or γ-globin; or the HSC contains or delivers a second HDR template. May be in contact with the particles or vector of. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier. In this regard, Cavazzana, "ex vivo lentiviral β ^A-T87Q - by globin vector of gene therapy of severe type β-thalassemia by implantation of autologous hematopoietic stem cells were transduced result (Outcomes of Gene Therapy for β- Thalassemia Major via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentivial β ^A-T87Q- Globin Vector) ". tif2014. org / abstractFiles / Jean% 20Antoine% 20Ribeil_Abstract. pdf; Cavazzana-Calvo, "Transfusion-independent and HMGA2 activation after gene therapy of human β-thalassemia (Transfusion independence and HMGA2 activation after gene therapy after gene therapy of human ) Doi: 10.1038 / nature09328; Nienhuis, "Development of Gene Therapy for Thalassemia", Cold Spring Harbor Perjectives. A 011833 (2012), LentiGlobin BB305, a lentiviral vector containing an engineered β-globin gene (β ^A-T87Q ); and Xie et al. , "Seamless gene mutation of β-thalassemia mutations in patient-specific iPSCsaging CRISPR / CRISPR / Cas9 and piggyback." 173427.114 (2014) http: // www. genome. org / cgi / doi / 10.1101 / gr. 173427.114 (Cold Spring Harbor Laboratory Press), which is the subject of Cavazzana's and Xie's research, including human β-thalassemia, both of which are referenced with all their references or related literature. To be incorporated herein by. In the present invention, the HDR template can provide an engineered β-globin gene (eg β ^A-T87Q ), or an HSC that expresses β-globin as it is in Xie.

Xu et al. (Sci Rep. 2015 Jul 9; 5: 12065. Doi: 10.1038 / rep12065) has designed TALEN and CRISPR-Cas9 to directly target the intron 2 mutation site IVS2-654 of the globin gene. .. Xu et al. Have observed different frequencies of double-strand breaks (DSBs) at the IVS2-654 locus using TALEN and CRISPR-Cas9, and TALEN is higher compared to CRISPR-Cas9 when combined with piggyBac transposon donors. Mediated homologous gene targeting efficiency. In addition, more obvious off-target events were observed in CRISPR-Cas9 compared to TALEN. Finally, TALEN-modified iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system, and relatively high HBB transcription was detected compared to unmodified cells.

Song et al. (Stem Cells Dev. 2015 May 1; 24 (9): 1053-65. Doi: 10.1089 / scd. 2014.0347. Epub 2015 Feb 5) modified β-Thal iPSC using CRISPR / Cas9. Human embryonic stem cells (hESCs) did not show off-target effects, so genetically modified cells exhibited normal nuclei and full pluripotency. Next, Song et al. Evaluated the differentiation efficiency of genetically modified β-Thal iPSC. Song et al. Found that during hematopoietic differentiation, genetically modified β-Thal iPSCs showed an increase in embryoid body ratio and proportion of various hematopoietic progenitor cells. More importantly, the genetically modified β-Thal iPSC strain restored HBB expression and reduced reactive oxygen species production compared to the unmodified group. Song et al. It was suggested that the hematopoietic differentiation efficiency of β-Thal iPSC was significantly improved when modified by the CRISPR-Cas9 system. Similar methods can be performed using the CRISPR-Cas9 system described herein, eg, a system containing a Cas9 effector protein.

WO 2015/148860 is cited, and through the teachings of this specification, the present invention includes methods and materials of these documents that apply in conjunction with the teachings of this specification. In certain aspects of blood-related disease gene therapy, methods and compositions for treating β-thalassemia may be adapted to the CRISPR-Cas system of the invention (see, eg, WO 2015/148860). In certain embodiments, WO 2015/148860 relates to the treatment or prevention of β-thalassemia, or its symptoms, eg, by altering the gene for B cell CLL / lymphoma 11A (BCL11A). The BCL11A gene is also known as B cell CLL / lymphoma 11A, BCL11A-L, BCL11A-S, BCL11AXL, CTIP1, HBFQTL5 and ZNF. BCL11A encodes a zinc finger protein involved in the regulation of globin gene expression. Altering the BCL11A gene (eg, one or both alleles of the BCL11A gene) can increase γ-globin levels. γ-globin can replace β-globin in the hemoglobin complex to effectively transport oxygen to tissues, which can improve the β-thalassemia disease phenotype.

Sickle cell anemia is an autosomal recessive disorder in which red blood cells become sickle-shaped. This is caused by a single nucleotide substitution of the β-globin gene located on the short arm of chromosome 11. As a result, valine is produced instead of glutamic acid, causing the production of sickle hemoglobin (HbS). As a result, distorted red blood cells are formed. This abnormal shape can occlude microvessels and cause serious damage to bone, spleen and skin tissue. This can lead to episodes of pain, frequent infections, hand-foot syndrome or even multiple organ failure. Distorted red blood cells are also prone to hemolysis, which can lead to severe anemia. As with β-thalassemia, sickle cell anemia can be corrected by modifying the HSC with the CRISPR-Cas9 system. This system is capable of specifically editing the genomic of a cell by cutting the DNA of the genome and then self-repairing it. When the Cas9 protein is inserted and guided by the RNA guide to the mutated site, the DNA is then cleaved at that site. At the same time, a healthy sequence is inserted. This sequence is used by the cell's self-repairing system to correct the induced cleavage. In this way, CRISPR-Cas9 is capable of correcting mutations in pre-obtained stem cells. Based on their knowledge in the art and the teachings of the present disclosure, the CRISPR-Cas9 system (eg, β-globin, preferably non-erythrocyte sickle) is used to target and correct mutations in sickle cell anemia. HSCs can be modified using suitable HDR templates that deliver the coding sequence for β-globin; specifically, guide RNAs target mutations that give rise to sickle cell anemia. And HDR can result in proper coding of β-globin expression. The mutated HSC is contacted with a guide RNA that targets particles containing the mutated-and-Cas9 proteins. The particles may also contain an HDR template suitable for correcting the mutation for proper expression of β-globin; or the HSC is a second particle or vector containing or delivering the HDR template. May be in contact with. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier. The HDR template can provide HSC to express the engineered β-globin gene (eg βA-T87Q), or β-globin as it is in Xie.

Pamphlets of WO 2015/148863 are also mentioned, and through the teachings of this specification, the present invention includes the methods and materials of these documents that can be applied to the CRISPR-Cas system of the present invention. In some embodiments of the treatment and prevention of the hereditary blood disease sickle cell disease, WO 2015/148863 pamphlet comprises altering the BCL11A gene. Altering the BCL11A gene (eg, one or both alleles of the BCL11A gene) can increase γ-globin levels. γ-globin can replace β-globin in the hemoglobin complex to effectively transport oxygen to tissues, which can improve the sickle cell disease phenotype.

Williams, "Broadening the Indications for Hematopoietic Stem Cell Genetic Therapies," Cell Stem Cell 13: 263-264 (2013), all shown with reference. (Incorporated herein) is an HSC / P from a patient with heterozygous white dystrophy (MLD) with lithosome storage disease, a hereditary disorder caused by deficiency of arylsulfatase A (ARSA) that causes neurodemyelination. Lentivirus-mediated gene transfer into cells; and patients with Viscott-Oldrich syndrome (WAS) (having a defect in the WAS protein, an effector of the small GTPase CDC42 that regulates cytoskeletal function in the blood cell lineage, and thus repeating Lentivirus-mediated genes for HSCs in patients suffering from immunodeficiency with infection, autoimmune symptoms, and thrombocytopenia with abnormally small dysfunctional platelets that result in high bleeding and increased risk of leukemia and lymphoma) Introductory has been reported; specifically, guide RNAs can target mutations that give rise to MLD (deficient ARSA), and HDR can provide coding for proper expression of ARSA. .. The mutated HSC is contacted with a guide RNA that targets particles containing the mutated-and-Cas9 proteins. The particles may also contain an HDR template suitable for correcting the mutation for proper expression of ARSA; or the HSC is in contact with a second particle or vector containing or delivering the HDR template. You may let me. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier. In contrast to the use of lentivirus, those skilled in the art have a mutation (arylsulfatase A (ARSA) deficiency) with respect to MLD (arylsulfatase A (ARSA) deficiency) based on knowledge in the art and teachings of the present disclosure. ) Can be modified using a CRISPR-Cas9 system (eg, including a suitable HDR template that delivers the coding sequence for ARSA). In contrast to the use of lentivirus, those skilled in the art will, based on their knowledge in the art and the teachings of the present disclosure, target and modify mutations (defects in the WAS protein) with respect to the CRISPR-Cas9 system (eg, eg). HSCs can be modified using a suitable HDR template that delivers the coding sequence for the WAS protein; specifically, the guide RNA targets mutations that give rise to WAS (deficient WAS protein). And HDR can result in proper coding of WAS protein expression. A guide RNA that targets the mutant-and-Cas9 protein-containing particles is contacted with the mutated HSC. The particles can also contain an HDR template suitable for modifying the mutation for proper expression of the WAS protein; or the HSC is contacted with a second particle or vector that contains or delivers the HDR template. You may. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier.

In certain aspects of the invention, by adapting the CRISPR-Cas system of the invention, methods and compositions involving editing of the target nucleic acid sequence, or regulation of expression of the target nucleic acid sequence, and its application in connection with cancer immunotherapy. Is included. Because one or more changes in one or more T cell expression genes, such as FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC and / or TRBC genes, affect T cell proliferation, survival and / or function. See the application of gene therapy in Pamphlet International Publication No. 2015/161276 with methods and compositions that can be used in. In a related embodiment, T cell proliferation is associated with one or more T cell expression genes, such as the CBLB and / or PTPN6 gene, FAS and / or BID gene, CTLA4 and / or PDCDI and / or TRAC and / or TRBC gene. Can be affected by change.

Chimeric antigen receptor (CAR) 19 T cells exhibit anti-leukemic effects in malignant lesions of patients. However, leukemia patients often do not have enough T cells to collect, which means that treatment should include modified T cells from the donor. Therefore, building a donor T cell bank is beneficial. Qasim et al. ("First Clinical Application of Tale Enginered Universal CAR19 T Cells in B-ALL" ASH 57th Ann. 2015, Abstract 2046 (https://ash.confex.com/ash/2015/webprogram/Paper81653.html online publication November 2015) eliminates graft-versus-host disease risk by disrupting T cell receptor expression and CD52 targeting In addition, CD52 cells are targeted to be insensitive to alemtuzumab, which in turn is host-mediated by human leukocyte antigen (HLA) mismatch CAR19 T cells. It became possible to prevent rejection. Researchers used a 3rd generation self-inactivating lentivirus vector encoding 4g7 CAR19 (CD19 scFv-4-1BB-CD3ζ) linked to RQR8, followed by Two pairs of TALEN mRNAs for multiple targeting to both the T cell receptor (TCR) α constant chain locus and the CD52 locus were electroperforated into the cells. CliniMacs α cells still expressing TCR after exobibo expansion. Depletion using / βTCR depletion resulted in <1% TCR expressing T cell product (UCART19), of which 85% expressed CAR19 and 64% became CD52 negative. Of this modified CAR19 T cell. Administration treated a patient with recurrent acute lymphoblastic leukemia. The teachings provided herein provide an effective method for modifying cells, eg, removing or regulating CD52 or other targets. It can be provided and thus used in conjunction with modifications of administration of T cells or other cells to the patient to treat malignant lesions.

Watts, "Hematopoietic Stem Cell Expansion and Gene Therapy" Cytotherapy 13 (10): 1164-1171. doi: 10.3109 / 14653249.2011.620748 (2011) (incorporated herein by reference as all indicated together with its references) is a hematological condition, immunity including HIV / AIDS Insufficiency and other genetic disorders such as lithosome storage, such as SCID-X1, ADA-SCID, β-salasemia, X-linked CGD, Viscott-Oldrich syndrome, Funcony anemia, adrenoleukodystrophy (ALD), Hematopoietic stem cell (HSC) gene therapy, such as virus-mediated HSC gene therapy, is being considered as a highly attractive treatment option for many disorders, including adrenoleukodystrophy (MLD).

US Patent Application Publication No. 20110225644, No. 20110091441, No. 2010222952, No. 20090271881 and No. 20090222937 transferred to Receptors are hereby relating to CREI variants. Then, at least one of the two I-CreI monomers is one in each of the two functional subdomains of the LAGLIDADG core domain located at positions 26-40 and 44-77 of I-CreI, respectively. Each having at least two substitutions, said mutant can cleave a DNA target sequence from the human interleukin-2 receptor γ chain (IL2RG) gene, also called the common cytokine receptor γ chain gene or γC gene. .. Nucleic acid targeting of the present invention by targeting the target sequences identified in U.S. Patent Application Publication No. 20110225664, 20110921441, 2010222252, 20090271881 and 20090222937. It can be used for the system.

Severe combined immunodeficiency disease (SCID) is caused by a defect in lymphocyte T maturation that is always accompanied by a functional defect in lymphocyte B (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585). 602; Fisher et al., Immunol. Rev., 2005, 203, 98-109). The overall incidence is estimated to be 1 in 75,000 live births. Untreated SCID patients are susceptible to multiple opportunistic microbial infections and generally do not live for more than a year. SCID can be treated by allogeneic hematopoietic stem cell transplantation from a family donor. Histocompatibility with donors can vary widely. In the case of adenosine deaminase (ADA) deficiency, which is one of the SCID forms, the patient can be treated by injection of recombinant adenosine deaminase enzyme.

Since it was revealed that the ADA gene is mutated in SCID patients (Giblett et al., Lancet, 1972, 2, 1067-1069), several other genes involved in SCID have been identified (Cavazzana). -Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fisher et al., Immunol. Rev., 2005, 203, 98-109). There are four major causes of SCID: (i) The most frequent form of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by mutations in the IL2RG gene, mature T lymphocytes and NK. The cells are no longer present. IL2RG encodes a γC protein, a component common to at least five interleukin receptor complexes (Noguchi, et al., Cell, 1993, 73, 147-157). These receptors activate several targets via JAK3 kinase (Macchi et al., Nature, 1995, 377, 65-68), the inactivation of which leads to the same syndrome as γC inactivation; ii) Mutation of the ADA gene results in a fatal deficiency of purine metabolism for lymphocyte progenitor cells, resulting in the near absence of B, T and NK cells; (iii) V (D) J recombination , An essential step in the maturation of immunoglobulins and T lymphocyte receptors (TCRs). Mutations in the three genes involved in this process, recombinant activation genes 1 and 2 (RAG1 and RAG2) and Artemis, result in a lack of mature T and B lymphocytes; and (iv) CD45 and other T cell-specific Mutations in other genes involved in target signaling have also been reported, but they correspond to a small number of cases (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fisher et al., Immunol. Rev., 2005, 203, 98-109). Since their genetic basis has been identified, these various SCID morphologies have been paradigms for gene therapy techniques for two main reasons (Fisher et al., Immunol. Rev., 2005, 203, 98-109). ). First, as with any blood disorder, exobibo treatment can be envisioned. Hematopoietic stem cells (HSCs) can be recovered from the bone marrow and retain their pluripotency properties over several cell divisions. Therefore, HSCs can be treated in vitro and then reinjected into the patient, and HSCs repopulate in the bone marrow. Second, modified cells have a selective advantage because lymphocyte maturation is impaired in SCID patients. Therefore, a small number of modified cells can restore the functional immune system. This hypothesis is based on the following assumptions: (i) Partial recovery of immune function associated with mutation recovery in SCID patients (Hirschhorn et al., Nat. Genet., 1996, 13, 290-295; Stephan et al., N. Engl. J. Med., 1996, 335, 1563-1567; Bousso et al., Proc. Natl., Acad. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl. Acad. Sci. USA, 2001,98,8697-8702; Nishikomori et al., Blood, 2004,103,4565-4572), (ii) Correction of SCID-X1 deficiency in vitro in hematopoietic cells (Candotti et al., Blood, 1996) , 87, 3097-3102; Cavazzana-Calvo et al., Blood, 1996, Blood, 88, 3901-3909; Taylor et al., Blood, 1996, 87, 3103-3107; Hacein-Bay et al., Blood, 1998, 92, 4090-4097), (iii) In vivo SCID-X1 (Soudais et al., Blood, 2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72- in animal models. 79), JAK-3 (Bunting et al., Nat. Med., 1998, 4,58-64; Bunting et al., Hum. Gene Ther., 2000, 11,235-2364) and RAG2 (Yates et al.) , Blood, 2002, 100, 3942-3949) by correction of deficiency, and (iv) by the results of gene therapy clinical trials (Cavazzana-Calvo et al., Science, 2000, 288, 669-672; AIuti et al. , Nat. Med., 2002; 8,423-425; Gaspar et al., Ranchet, 2004,364,2181-2187), verified several times.

US Pat. It relates to a method and use for adjusting HbF). Targets disclosed in US Patent Application Publication No. 20110182867, such as BCL11A, can be targeted by the CRISPR Cas9 system of the invention to regulate fetal hemoglobin expression. For further BCL11A targets, see Bauer et al. (Science 11 October 2013: Vol. 342 no. 6155 pp. 253-257) and Xu et al. See also (Science 18 November 2011: Vol. 334 no. 6058 pp. 993-996).

Those skilled in the art can modify HSCs for genetic hematological disorders such as β-thalassemia, hemophilia, or genetic lysosomal storage disease, based on their knowledge in the art and the teachings of the present disclosure.

Treatment of Brain, Central Nervous System and Immune System Diseases The present invention also contemplates delivering the CRISPR-Cas system to the brain or neurons. For example, RNA interference (RNAi) provides therapeutic potential for this disorder by reducing the expression of HTT, the disease-causing gene for Huntington's disease (eg, McBride et al., Molecular Therapy vol. 19 no. 12). (See Dec. 2011, pp. 2152-2162), therefore the applicant assumes that it can be used and / or adapted to the CRISPR-Cas system. The CRISPR-Cas system can be generated using algorithms that reduce the likelihood of off-targeting of the antisense sequence. The CRISPR-Cas sequence targets any sequence in exon 52 of mouse, rhesus monkey or human huntingtin and can be expressed in viral vectors such as AAV. Approximately 3 microinjections per hemisphere (6 injections total) to animals, including humans: first 1 mm rostral (12 μl) from the anterior commissure and the remaining 2 injections (12 μl and 10 μl, respectively) first It may be injected 3 and 6 mm caudally from the injection at a rate of about 1 μl / min by 1e12 vg / ml AAV, leaving the needle in place for an additional 5 minutes to diffuse the infusion solution from the needle tip. Was done.

DiFiglia et al. (PNAS, October 23, 2007, vol. 104, no. 43, 17204-17209), a single dose of siRNA targeting Htt to the adult striatum silences the mutant Htt and causes neurological lesions. It was observed that the abnormal behavioral phenotype observed in the acute-onset viral transgenic mouse HD model could be delayed. DiFiglia injected 2 μl of Cy3-labeled cc-siRNA-Htt or non-conjugated siRNA-Htt into mice at 10 μM intrastripe. Similar dosages of Htt-targeted CRISPR Cas can be contemplated in humans in the present invention, for example, about 5-10 ml of Htt-targeted 10 μM CRISPR Cas may be infused intrastripe. ..

In another example, Boudriau et al. (Molecular Therapy vol.17 no.6 june 2009) injects 5 μl of recombinant AAV serotype 2/1 vector expressing the http-specific RNAi virus into the striatum (at 4 × 1012 viral genome / ml). .. Similar dosages of Htt-targeted CRISPR Cas can be contemplated in humans in the present invention, eg, about 10-20 ml of Htt-targeted 4 × 10 ¹² viral genome / ml) CRISPR Cas. It may be injected into the striatum.

In another example, HTT-targeted CRISPR Cas can be administered continuously (see, eg, Yu et al., Cell 150, 895-908, August 31, 2012). Yu et al. Delivered 300 mg / day of ss-siRNA or phosphate buffered saline (PBS) (Sigma-Aldrich) for 28 days using an osmotic pump (model 2004) delivering 0.25 ml / hour and 0. A positive control MOE ASO of 75 mg / day was delivered for 14 days using a pump (model 2002) designed to deliver .5 μl / hour. The pump (Durect Corporation) was filled with diluted ss-siRNA or MOE in sterile PBS and then incubated at 37 ° C. 24 or 48 hours before implantation (model 2004). Mice were anesthetized with 2.5% isoflurane and a midline incision was made at the base of the skull. A cannula was implanted in the right lateral ventricle using a stereotaxic guide and fixed with Loctite adhesive. A catheter attached to the Alzet osmotic minipump was attached to the cannula and the pump was placed subcutaneously in the central region of the scapula. The incision was closed with 5.0 nylon suture. Similar dosages of Htt-targeted CRISPR Cas can be contemplated in humans in the present invention, for example, about 500-1000 g / day of Htt-targeted CRISPR Cas may be administered.

In another example of continuous infusion, Stiles et al. (Experimental Neurology 233 (2012) 463-471) implanted a parenchymal catheter with a titanium needle tip into the right putamen. A catheter was connected to a SynchromMed® II pump (Medtronic Neurological, Minneapolis, MN) implanted subcutaneously in the abdomen. After injecting phosphate buffered saline at 6 μL / day for 7 days, the pump was refilled with the test substance and 7 days of continuous delivery was programmed. Approximately 2.3 to 11.52 mg / day of siRNA was injected at various injection rates of approximately 0.1 to 0.5 μL / min. Similar dosages of Htt-targeted CRISPR Cas can be contemplated in humans in the present invention, for example, about 20-200 mg / day of Htt-targeted CRISPR Cas may be administered. In another example, the method of U.S. Patent Application Publication No. 20130253040 transferred to Sangamo (Pamphlet International Publication No. 2013130824) is also adapted from TALES to the CRISPR Cas system of the invention for the treatment of Huntington's disease. Can be done. Broad Institute et al. Pamphlet 2015089354 A1 (incorporated by reference herein) in the name of Huntington's Disease (HP) describes the target of Huntington's disease (HP). Possible target genes for the CRISPR complex for Huntington's disease: PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; and TGM2.

Therefore, one or more of PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; and TGM2 may be selected as targets for Huntington's disease in some embodiments of the invention.

Other trinucleotide repeat disorders. These may include one of the following: Category I includes Huntington's disease (HD) and spinocerebellar ataxia; Category II elongation is phenotypically diverse and generally small in scale. With heterogeneous dilation also found in gene exons; and category III includes fragile X syndrome, muscular tonic dystrophy, two of spinocerebellar ataxia, juvenile myoclonus epilepsy, Friedreich's ataxia. ..

A further aspect of the invention relates to the use of the CRISPR-Cas system to correct defects in the EMP2A and EMP2B genes that have been identified as being associated with Lafora disease. Lafora disease is an autosomal recessive condition characterized by progressive myoclonic epilepsy that can begin as a seizure in adolescence. Several cases of this disease can be caused by mutations in genes that have not yet been identified. The disease causes seizures, muscle cramps, difficulty walking, dementia, and ultimately death. Currently, there are no treatments that have proven effective for disease progression. Other genetic abnormalities associated with epilepsy can also be targeted by the CRISPR-Cas system, the underlying genetics of which are Genetics of Epilepsy and Genetic Epilepsy, editor Giuliano Avanzani, Jeffrey L. et al. It is further described in Noevels, Mariani Foundation Pediatric Neurology: 20; 2009).

Sangamo BioSciences, Inc. on inactivating the T cell receptor (TCR) gene. The method of U.S. Patent Application Publication No. 201101158957, assigned to CRISPR Cas, can also be modified for the CRISPR Cas system of the invention. In another example, Sangamo BioSciences, Inc., both relating to inactivating the glutamine synthetase gene expression gene. The methods of U.S. Patent Application Publication No. 201011124 assigned to CRESPRIS and U.S. Patent Application Publication No. 20110225664 assigned to Celtecis may also be modified for the CRISPR Cas system of the invention.

Treatment of Hearing Impairment The present invention also contemplates delivering the CRISPR-Cas system to one or both ears.

Researchers are investigating whether gene therapy can be used to assist with current hearing loss treatments, cochlear implants. Hearing loss is often caused by the loss or damage of hair cells that prevent signals from being relayed to auditory neurons. In that case, a cochlear implant can be used to respond to sound and transmit electrical signals to nerve cells. However, these neurons are often degenerated and receding from the cochlea with reduced release of growth factors by impaired hair.

U.S. Patent Application Publication No. 20120328580 describes the injection of a pharmaceutical composition into the ear (eg, auricular administration) using a syringe, eg, a single dose syringe, eg, the tympanic cavity (eg, the central floor, etc.). The injection to the vestibular floor and scala tympani floor is described. For example, one or more of the compounds described herein can be administered by intratympanic injection (eg, into the middle ear) and / or by injection into the outer, middle, and / or inner ear. Such methods are commonly used in the art, for example, for the administration of steroids and antibiotics to the human ear. The infusion may be, for example, through the round window of the ear or through the cochlear sac. Other methods of inner ear administration are known in the art (see, eg, Salt and Plantke, Drug Discovery Today, 10: 1299-1306, 2005).

In another method of administration, the pharmaceutical composition can be administered in situ using a catheter or pump. The catheter or pump can, for example, pump the pharmaceutical composition into the cochlear duct or the round window of the ear and / or the lumen of the colon. An exemplary drug delivery device and method suitable for administering one or more of the compounds described herein to the ear, eg, the human ear, is described by McKenna et al. (US Patent Application Publication No. 2006/0030837) and Jacobsen et al. (US Pat. No. 7,206,639). In some embodiments, the catheter or pump may be placed, for example, in the patient's ear (eg, the outer ear, middle ear, and / or inner ear) during the surgical procedure. In some embodiments, the catheter or pump can be placed, for example, in the patient's ear (eg, outer ear, middle ear, and / or inner ear) without the need for surgical procedures.

Alternatively or additionally, one or more of the compounds described herein can be administered in combination with a mechanical device worn on the outer ear, such as a cochlear implant or hearing aid. An exemplary cochlear implant suitable for use in the present invention is described in Edge et al. (US Patent Application Publication No. 2007/093878).

In some embodiments, the administration methods described above may be combined in any order and may be simultaneous or dispersed.

Alternatively or additionally, the present invention is described in, for example, CDER Data Standards Manual, 004th Edition (available at fda.give/cder/dsm/DRG/drg00301.htm), Food and Drug Administration (Food). It may be administered according to any of the methods approved by and Drug Administration).

In general, using the cell therapy methods described in U.S. Patent Application Publication No. 20120328580, in vitro to or to the mature cell type of the inner ear (eg, hair cells) or to the cell, complete or partial. Differentiation can be promoted. The cells obtained by such a method can be transplanted or implanted in a patient in need of the next such treatment. For the cell culture methods required to carry out such methods, suitable cell type identification and selection methods, methods for promoting complete or partial differentiation of selected cells, complete or partial differentiation. The methods for identifying the cell type and the method for implanting fully or partially differentiated cells are described below.

Suitable cells for use in the present invention are, but are not limited to, mature cells of the inner ear, such as hair cells, when contacted with one or more of the compounds described herein, eg, in vitro. For example, cells having the ability to completely or partially differentiate into inner hair cells and / or outer hair cells). Exemplary cells capable of differentiating into hairy cells include, but are not limited to, stem cells (eg, inner ear stem cells, adult stem cells, bone marrow-derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS cells, and Fat-derived stem cells), progenitor cells (eg, inner ear progenitor cells), supporting cells (eg, ditelus cells, pillar cells, inner phalangeal cells, optic tectum cells and Henzen cells), and / or germ cells. The use of stem cells to replenish inner ear sensory cells is described in Li et al. (US Patent Application Publication No. 2005/0287127) and Li et al. (US Patent Application No. 11 / 953,797). The use of bone marrow-derived stem cells to replace inner ear sensory cells is described in Edge et al. , PCT / US Patent Application Publication No. 2007/084654. For iPS cells, for example, Takahashi et al. , Cell, Volume 131, Issue 5, Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006); Okita et al. , Nature 448, 260-262 (2007); Yu, J. et al. et al. , Science 318 (5858): 1917-1920 (2007); Nakagawa et al. , Nat. Biotechnol. 26: 101-106 (2008); and Zaehres and Scholar, Cell 131 (5): 834-835 (2007). Such suitable cells can be identified by (eg, qualitatively or quantitatively) analyzing the presence of one or more tissue-specific genes. For example, gene expression can be detected by detecting a protein product of one or more tissue-specific genes. Protein detection techniques include staining proteins (eg, using cell extracts or whole cells) with antibodies against the appropriate antigen. In this case, the suitable antigen is a protein product of tissue-specific gene expression. In principle, a primary antibody (ie, an antibody that binds to an antigen) can be labeled, but it is more common (and improved visualization) to use a secondary antibody that targets the primary antibody (eg, anti-IgG). To do). This secondary antibody is conjugated to a fluorescent dye or to a colorimetric reaction with a suitable enzyme, or to gold beads (for electron microscopy), or a biotin-avidin system, thereby primary. It becomes possible to recognize the position of an antibody and thus an antigen.

The CRISPR Cas molecule of the present invention can be delivered to the ear by applying the pharmaceutical composition directly to the outer ear by a composition modified from US Patent Application Publication No. 201101142917. In some embodiments, the pharmaceutical composition is applied to the ear canal. Delivery to the ear may also be referred to as aural delivery or otic delivery.

In some embodiments, the RNA molecule of the invention is delivered in a liposome formulation, lipofectin formulation, etc. and can be prepared by methods well known to those of skill in the art. Such methods include, for example, US Pat. Nos. 5,593,972, 5,589,466 and 5,580,859, which are incorporated herein by reference. )It is described in.

Delivery systems have been developed specifically for enhancing and improving the delivery of siRNA to mammalian cells (eg, Shen et al FEBS Let. 2003, 539: 111-114; Xia et al., Nat. Biotech. 2002). , 20: 1006-1010; Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis et al., Nat. See Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11: 2717-2724), which can be applied to the present invention. SiRNA has been successfully used in primates to suppress gene expression in recent years (see, eg, Tolentino et al., Retina 24 (4): 660, which can also be applied to the present invention. ).

Qi et al. Discloses an efficient siRNA transfection method for the inner ear through an intact round window with a novel protein delivery technique, which can be applied to the CRISPR Cas system of the invention (eg, Qi et). al., Gene Therapy (2013), 1-9). Specifically, the TAT double-stranded RNA-binding domain (TAT-DRBD) is found in cells of the inner ear (eg, inner and outer hair cells), ampulla ridges, macula and macula. Cy3-labeled siRNA can be transfected by permeation through an intact round window), successful in vivo delivery of double-stranded siRNA for treating various inner ear disorders and maintaining auditory function. ing. Approximately 40 μl of 10 mM RNA can be contemplated as a dosage for administration to the ear.

Rejari et al. According to (Hear Res. 2007 Jun; 228 (1-2): 180-7), good maintenance of spiral ganglion neurons, which are the targets of electrical stimulation by implants, can improve cochlear implant function. Brain-derived neurotrophic factors (BDNF) have previously been shown to enhance spiral ganglion survival in deafened ears. Rejari et al. Tested a modified design cochlear implant electrode containing a coating of fibroblasts transduced by a viral vector carrying a BDNF gene insert. To achieve this type of ex vivo gene transfer, Rejari et al. Transduced adenovirus with BDNF gene cassette inserts into guinea pig fibroblasts, determined that these cells secreted BDNF, and then attached the BDNF-secreting cells to the artificial inner ear electrode with an agarose gel. The electrodes were implanted on the scala tympani floor. Rejari et al. Determined that this BDNF-expressing electrode was able to maintain significantly more spiral ganglion neurons in the basal rotation of the cochlea when compared to control electrodes 48 days after implantation, and found that the spiral ganglion neurons of We have demonstrated the feasibility of combining cochlear implant therapy with exvivo gene transfer to enhance survival. Such a system can be applied to the delivery of the nucleic acid targeting system of the present invention to the ear.

Mukherjea et al. (Antioxidants & Redox Signaling, Volume 13, Number 5,2010) are NOX3 using small interfering (si) RNA, as evidenced by protection of OHC from injury and reduced threshold shift in auditory brainstem response (ABR). Knockdown reports that cisplatin addictive hearing loss has been resolved. Various doses of siNOX3 (0.3, 0.6, and 0.9 μg) were administered to rats and NOX3 expression was assessed by real-time RT-PCR. The lowest dose (0.3 μg) of NOX3 siRNA used did not show any inhibition of NOX3 mRNA when compared to scrambled siRNA or transtympanic administration of untreated cochlea. However, administration of higher doses of NOX3 siRNA (0.6 and 0.9 μg) reduced NOX3 expression compared to control scrambled siRNA. Such a system can be applied to the CRISPR Cas system of the present invention for transtympanic administration with a dosage of about 2 mg to about 4 mg of CRISPR Cas for administration to humans.

Jung et al. (Molecular Therapy, vol.21 no. 4,834-841 appr. 2013) showed that Hes5 levels in the utricles decreased after application of siRNA, and the number of hair cells in those utricles was controlled. It demonstrates that it was significantly higher than later. This data suggests that siRNA techniques may be useful in inducing repair and regeneration in the inner ear, and that the Notch signaling pathway is a potentially useful target for inhibition of specific gene expression. Jung et al. Infused into the vestibular epithelium of the ear a 2 μl volume of 8 μg Hes5 siRNA prepared by adding sterile normal saline to the lyophilized siRNA. Such a system can be applied to the CRISPR Cas system of the invention for administration to the vestibular epithelium of the ear with a dosage of about 1 to about 30 mg of CRISPR Cas for administration to humans.

Treatment of Eye Diseases The present invention also contemplates delivering the CRISPR-Cas9 system to one or both eyes.

In yet another aspect of the invention, using the CRISPR-Cas9 system, Genetic Diseases of the Eye, Second Edition, editor Elias I. et al. Eye abnormalities caused by some gene mutations further described in Traboulsi, Oxford University Press, 2012 can be corrected.

Lentiviral vectors, specifically equine infectious anemia virus (EIAV), are particularly preferred for ocular administration.

In another embodiment, a minimal non-primate lentiviral vector based on equine infectious anemia virus (EIAV) is also contemplated, especially for gene therapy of the eye (eg, Balagaan, J Gene Med 2006; 8). : 275-285, published online 21 November 2005, Wiley InterScience (see www.interscience.virus.com). DOI: 10.1002 / jgm.845). The vector is intended to have a cytomegalovirus (CMV) promoter that drives the expression of the target gene. Intra-anterior, sub-retinal, intraocular and intravitreal injections are all intended (eg, Balagaan, J Gene Med 2006; 8: 275-285, published online 21 November 2005, Wiley InterScience (www.interscience.wiley.com). ). DOI: 10.10012 / jgm.845). Intraocular injection is performed with the help of a surgical microscope. For subretinal and intravitreal injections, lightly press with a finger to escape the eye and visualize the fundus using a contact lens system consisting of a drop of propagating medium solution on the cornea covered with a glass microscope slide cover slip. You may. For subretinal injection, the tip of a 10 mm 34 gauge needle attached to a 5 μl Hamilton syringe is directly visualized from the superior equatorial sclera tangentially toward the posterior pole until the needle hole is visible in the subretinal cavity. You may proceed. Next, 2 μl of vector suspension can be injected to cause upper retinal detachment, thus confirming subretinal vector administration. This technique creates a self-closing wound scleral incision, allowing the vector suspension to be maintained in the subretinal space until absorbed by the RPE within 48 hours of the normal procedure. This procedure may be repeated in the lower hemisphere to cause lower retinal detachment. By this technique, approximately 70% of the sensory nerve retina and RPE will be exposed to the vector suspension. For intravitreal injection, the tip of the needle may be advanced from the sclera 1 mm behind the corneal sclera and 2 μl of vector suspension may be injected into the vitreous cavity. For anterior chamber injection, the tip of the needle may be advanced by corneal scleral puncture, fed towards the central part of the cornea, and injected with 2 μl of vector suspension. For anterior chamber injection, the tip of the needle may be advanced by corneal scleral puncture, fed towards the central part of the cornea, and injected with 2 μl of vector suspension. These vectors can be injected with a titer of either 1.0 to 1.4 × 10 ¹⁰ or 1.0 to 1.4 × 10 ⁹ transduction units (TU) / ml.

In another embodiment, a horse-borne anemia virus-based lentivirus gene that expresses the angiogenesis-suppressing proteins endostatin and angiostatin delivered by subretinal injection for the treatment of moist age-related luteal degeneration. Therapeutic vector RetinoStat® is also contemplated (see, eg, Binley et al., HUMAN GENE THERAPY 23: 980-991 (September 2012)). Such a vector can be modified for the CRISPR-Cas9 system of the present invention. Each eye can be treated with a RetinoStat® with a dose of 1.1 × 10 ⁵ transduction units (TU / eye) and a total volume of 100 μl per eye.

In certain embodiments, WO 2015/153780 is cited, which comprises providing treatment or prevention of primary open-angle glaucoma (POAG) by targeting the coding sequence of the MYOC gene. To do. Some, but not limited to, target mutations that give rise to POAG are P370 (eg P370L); I477 (eg I477N or I477S); T377 (eg TE77R); Q368 (Q368 termination) (all in the MYOC gene). There is). Targeted mutations can also include mutation hotspots between amino acid sequence positions 246-252 of the MYOC gene. In certain embodiments, the target mutation is a mutation hotspot between the amino acid sequence positions of the MYOC gene, eg, amino acids 368-380, amino acids 368-370 + 377-380, amino acids 364-380, or amino acids 347-380. .. In certain embodiments, the target mutation is a mutation hotspot at amino acid sequence positions 423-437 of the MYOC gene (eg, amino acids 423-426, amino acids 423-427 and amino acids 423-437). In certain embodiments, the target mutation is a mutation hotspot located at amino acid sequence positions 477-502 of the MYOC gene (see, eg, WO 2015/153780).

In another embodiment, E1 deletion, partial E3 deletion, E4 deletion adenovirus vectors can be contemplated for delivery to the eye. E1 deletion, partial E3 deletion, E4 deletion adenovirus vector (AdPEDF.ll) expressing human pigment epithelial-derived factor in 28 patients with progressive neovascular age-related macular degeneration (AMD) Intravitreal injections were administered (see, eg, Campochiaro et al., Human Gene Therapy 17: 167-176 (February 2006)). Dose in the range of 106 to 109.5 particle units (PU) was examined and AdPEDF. There were no serious adverse events and dose limiting toxicities associated with ll (see, eg, Campochiaro et al., Human Gene Therapy 17: 167-176 (February 2006)). Adenovirus vector-mediated intraocular gene transfer appears to be a viable technique for the treatment of eye disorders and can be applied to the CRISPR Cas9 system.

In another embodiment, the RXi Pharmaceuticals sd-rxRNA® system can be used and / or adapted for delivery of CRISPR Cas9 to the eye. In this system, a single intravitreal administration of 3 μg of sd-rxRNA results in a sequence-specific reduction in PPIB mRNA levels over 14 days. The sd-rxRNA® system can be applied to the nucleic acid targeting systems of the invention with the intention of administering to humans a dose of about 3-20 mg of CRISPR.

Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4,642-649 appr. 2011) is resistant to inhibition due to RNA interference (RNAi) -based rhodopsin inhibitor and nucleotide changes in degenerate positions across RNAi target sites. Adeno-associated virus (AAV) vectors for delivering codon-modified rhodopsin substitution genes of. Injections of either 6.0 × 10 ⁸ vp or 1.8 × 10 ¹⁰ vp AAV can be performed at Millington-Ward et al. Was injected subretinal into the eye. Millington-Ward et al. The AAV vector of the above can be applied to the CRISPR Cas9 system of the present invention with the intention of administering to humans a dose of about 2 × 10 ¹¹ to about 6 × 10 ¹³ vp.

Dalkara et al. (Sci Transl Med 5,189ra76 (2013)) also relates to in vivo directed evolution that produces an AAV vector that delivers a wild-type version of the defective gene throughout the retina after non-injurious injection into the vitreous humor of the eye. Dalkara describes a 7mer peptide presentation library and an AAV library constructed by DNA shuffling of the cap genes AAV1, 2, 4, 5, 6, 8, and 9. These rcAAV libraries and the rAAV vector expressing GFP under the CAG or Rho promoter were packaged and quantitative PCR gave deoxyribonuclease resistant genomic titers. The libraries were pooled and two rounds of evolution were performed, each consisting of an initial library diversification followed by three in vivo selection steps. In each of these steps, P30 rho-GFP mice were intravitreally injected with 2 ml of iodixanol purified phosphate buffered saline (PBS) dialysis library with a genomic titer of approximately 1 × 10 ¹² vg / ml. Dalkara et al. The AAV vector of AAV vector can be applied to the nucleic acid targeting system of the present invention with the intention of administering to humans a dose of about 1 × 10 ¹⁵ to about 1 × 10 ¹⁶ vg / ml.

In another embodiment, the rhodopsin gene may be targeted for the treatment of retinitis pigmentosa (RP), where Sangamo BioSciences, Inc. The system of U.S. Patent Application Publication No. 20120204282 assigned to may be modified according to the CRISPR Cas9 system of the present invention.

In another embodiment, the method of US Patent Application Publication No. 20130183282 for a method of cleaving a target sequence from a human rhodopsin gene, assigned to Celectis, can also be modified for the nucleic acid targeting system of the invention.

US Patent Application Publication No. 20130202678, transferred to Academia Sinica, describes the Puf-A gene, which is expressed in retinal ganglion cells and pigment-containing cells of ophthalmic tissue, and exhibits unique anti-apoptotic activity. Concerning methods of treating retinopathy and vision-threatening ophthalmologic disorders relating to delivery to the subretinal or intravitreal space. In particular, the desired targets are zgc: 193333, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1 and HtrA2, all of which can be targeted by the nucleic acid targeting system of the present invention.

Wu (Cell Stem Cell, 13: 659-62, 2013) designed a guide RNA that leads Cas9 to a single base pair mutation that causes cataracts in mice, where Cas9 induced DNA cleavage. Either of the other wild-type alleles or oligos provided to the zygote repair mechanism were then used to correct the sequence of the broken allele in the mutant mouse and to correct the genetic defect that causes cataracts.

U.S. Patent Application Publication No. 20120159653 describes genetic alterations of cells, animals and proteins associated with zinc finger nucleases (MD). Macular degeneration (MD) is a major cause of visual impairment in the elderly, but is also a child with an onset age of infancy, such as Stargard's disease, Swordsby's fundus, and lethal pediatric neurodegenerative disease. It is also a prominent symptom of stage disease. Macular degeneration results in loss of vision in the center of the visual field (spots) due to damage to the retina. Current existing animal models do not reproduce the key features of the disease as observed in humans. Available animal models containing mutant genes encoding proteins associated with MD also develop highly variable phenotypes, making interpretation and therapeutic development for human disease difficult.

One aspect of US Patent Application Publication No. 20120159653 relates to editing any chromosomal sequence encoding a protein associated with MD, which may be applicable to the nucleic acid targeting system of the present invention. MD-related proteins are typically selected based on the experimental association of MD-related proteins with MD disorders. For example, a population with MD disorders may have increased or decreased production rates or circulating concentrations of proteins associated with MD as compared to populations without MD disorders. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, MD-related proteins are available, but not limited to, using genomics techniques including DNA microarray analysis, gene expression linkage analysis (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). It can be identified by obtaining the gene expression profile of the encoding gene.

Non-limiting examples of proteins associated with MD include, but are not limited to, the following proteins: (ABCA4) ATP binding cassette, subfamily A (ABC1), member 4 ACHM1 dyschromia (chemokine color vision). Abnormality) 1 ApoE Apolipoprotein E (ApoE) C1QTNF5 (CTRP5) C1q and tumor necrosis factor-related protein 5 (C1QTNF5) C2 complement component 2 (C2) C3 complement component (C3) CCL2 chemokine (CC motif) ligand 2 (CCL2) CCR2 Chemokine (CC motif) Receptor 2 (CCR2) CD36 Surface antigen classification 36 CFB Complement factor B CFH Complement factor CFH H CFHR1 Complement factor H-related 1 CFHR3 Complement factor H-related 3 CNGB3 Cyclic nucleotide Open Channel β3 CP Celluloplasmin (CP) CRP C Reactive Protein (CRP) CST3 Cystatin C or Cystatin 3 (CST3) CTSD Catepsin D (CTSD) CX3CR1 Chemokine (C-X3-C Motif) Receptor 1 ELOVL4 Extremely Long Chain Fatty Acid Elongation 4 ERCC6 Removal Repair Cross Complement Complement Repair Defect, Complement Group 6 FBRN5 Fiburin 5 FBRN5 Fiburin 5 FBRN6 Fiburin 6 FSCN2 Fascin (FSCN2) HMCN1 Chemokine (Hemicentrin) 1 HMCN1 Chemokine 1 HMCN1 Serin Peptidase 1 IL-6 Interleukin 6 IL-8 Interleukin 8 LOC387715 Virtual protein PLEKHA1 Plextrin homologous domain containing Family A member 1 (PLEKHA1) PROM1 Prominine 1 (PROM1 or CD133) PPPH2 Periferin-2 RPGR retinal pigment degeneration GTPase Regulators SERPING1 Serpin Peptidase Inhibitor, Clade G, Member 1 (C1-Inhibitor) TCOF1 Trickle TIMP3 Metalloproteinase Inhibitor 3 (TIMP3) TLR3 Toll-like Receptor 3.

The identities of MD-related proteins whose chromosomal sequences are edited can vary and vary. In a preferred embodiment, the MD-related protein whose chromosome sequence is edited is an ATP binding cassette encoded by the ABCR gene, a subfamily A (ABC1) member 4 protein (ABCA4), an apolypoprotein E protein encoded by the APOE gene (APOE gene). APOE), chemokine (CC motif) ligand 2 protein (CCL2) encoded by the CCL2 gene, chemokine (CC motif) receptor 2 protein (CCR2) encoded by the CCR2 gene, encoded by the CP gene It can be a celluloplasmin protein (CP), a catepsin D protein (CTSD) encoded by the CTSD gene, or a metalloproteinase inhibitor 3 protein (TIMP3) encoded by the TIMP3 gene. In an exemplary embodiment, the animal undergoing genetic modification is a rat, and the edited chromosomal sequence encoding an MD-related protein can be: (ABCA4) ATP binding cassette, NM_000350 subfamily A (ABC1), member. 4 APOE Apolipoprotein E NM_138828 (APOE) CCL2 Chemokine (C-C NM_031530 motif) Litogen 2 (CCL2) CCR2 Chemokine (CC NM_021866 motif) Receptor 2 (CCR2) CP Celluloplasmin (CP) NM_012532 CTSD ) NM_134334 TIMP3 metalloproteinase NM_012886 Inhibitor 3 (TIMP3). The animal or cell is 1, 2, 3, 4, 5, 6, 7 or more disrupted chromosomal sequences encoding MD-related proteins and 0, 1, 2, 2, encoding disrupted MD-related proteins. It may contain sequences integrated into 3, 4, 5, 6, 7 or more chromosomes.

The chromosomal sequence that is edited or integrated can be modified to encode a altered MD-related protein. Several mutations in MD-related chromosomal sequences have been associated with MD. Non-limiting examples of mutations in MD-related chromosomal sequences include E471K (ie, glutamic acid at position 471 is converted to lysine), R1129L (ie, arginine at position 1129 is converted to leucine), and T1428M (ie, arginine at position 1129) in the ABCR protein. That is, threonine at position 1428 is converted to methionine), R1517S (that is, arginine at position 1517 is converted to serine), I1562T (that is, isoleucine at position 1562 is converted to threonine), and G1578R (that is, glycine at position 1578 is converted to arginine). V64I in CCR2 protein (ie, valine at position 192 is converted to isoleucine); G969B in CP protein (ie, glycine at position 969 is converted to aspartic acid or aspartic acid); S156C in TIMP3 protein (ie, serine at position 156) G166C (ie, glycine at position 166 changes to cysteine), G167C (ie, glycine at position 167 changes to cysteine), Y168C (ie, tyrosine at position 168 changes to cysteine), S170C (ie, changes to cysteine), S170C (ie, changes to cysteine) Those that cause MD include those that cause MD, including Y172C (ie, tyrosine at position 172 is converted to cysteine) and S181C (ie, serine at position 181 is converted to cysteine). Other associations of MD-related genes and genetic variation in disease are known in the art.

Treatment of Cardiovascular and Muscle Diseases The present invention also contemplates delivering the CRISPR-Cas system described herein, eg, the Cas9 effector protein system, to the heart. For the heart, myocardial adeno-associated virus (AAVM), specifically AAVM41, which has shown preferential gene transfer in the heart, is preferred (eg, Lin-Yanga et al., PNAS, March 10, 2009). , Vol. 106, no. 10). Administration may be systemic or topical. A dosage of about 1-10 × 10 ¹⁴ vector genome is intended for systemic administration. For example, Eulario et al. (2012) Nature 492: 376 and Somasuntharam et al. (2013) See also Biomaterials 34: 7790.

For example, US Patent Application Publication No. 20110023139 describes genetic alterations of cells, animals and proteins associated with cardiovascular disease using zinc finger nucleases. Cardiovascular disease generally includes hypertension, heart attack, heart failure, and stroke and TIA. In the methods described in this disclosure, proteins encoded by any chromosomal sequence involved in cardiovascular disease or any chromosomal sequence involved in cardiovascular disease may be utilized. Cardiovascular-related proteins are typically selected based on the experimental association between cardiovascular-related proteins and the development of cardiovascular disease. For example, populations with cardiovascular disorders may have increased or decreased production rates or circulating concentrations of cardiovascular-related proteins compared to populations without cardiovascular disorders. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, cardiovascular-related proteins may be encoded using genomics techniques including, but not limited to, DNA microarray analysis, gene expression linkage analysis (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). It can be identified by obtaining the gene expression profile of the gene.

As an example, the chromosome sequence is not limited, but IL1B (interleukin 1, β), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostacyclin 12 (prostacyclin) synthase), MB (myoglobin). ), IL4 (interleukin 4), ANGPT1 (angiopoetin 1), ABCG8 (ATP binding cassette, subfamily G (WHITE), member 8), CTSK (catepsin K), PTGIR (prostacyclin 12) Receptor (IP)), KCNJ11 (potassium inward rectifying channel, subfamily J, member 11), INS (insulin), CRP (C-reactive protein, pentraxin-related), PDGFRB (platelet-derived growth factor receptor, β Polypeptide), CCNA2 (cyclin A2), PDGFB (platelet-derived growth factor β-polypeptide (monkey for monkey sarcoma virus (v-sis) cancer gene homologue)), KCNJ5 (potassium inward rectifying channel, subfamily J, member 5) , KCNN3 (potassium intermediate / small conductance calcium activation channel, subfamily N, member 3), CAPN10 (calpine 10), PTGES (prostacyclin E synthase), ADRA2B (adrenalinergic, α-2B-, receptor) ), ABCG5 (ATP binding cassette, subfamily G (WHITE), member 5), PRDX2 (peroxyredoxin 2), CAPN5 (calpine 5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C ( mex-3 homolog C (C. elegance)), ACE angiotensin I-converting enzyme (peptidyl dipeptidase A) 1), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interlegans) 6 (interferon, β2)), STN (statin), SERPINE1 (cellpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin, C1Q and Collagen domain included), APOB (Apolipoprotein B (including Ag (x) antigen)), APOE (Apolipoprotein E), LEP (Leptin), MTHR (5,10-methylenete) Trahydrofolate reductase (NADPH)), APOA1 (apolypoprotein AI), EDN1 (endoserine 1), NPBP (natriuretic peptide precursor B), NOS3 (nitrogen monoxide synthase 3 (endothelial cells)), PPARG ( Peroxysome growth factor activated receptor γ), PLAT (plasminogen activator, tissue), PTGS2 (prostaglandin endoperoxide synthase 2 (prostaglandin G / H synthase and cyclooxygenase)), CETP (cholesteryl ester transfer protein, plasma ), AGTR1 (angiotensin II receptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE (selectin E) , REN (renin), PPARA (peroxysome growth factor activated receptor α), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2 (chemocain (CC motif) ligand 2), LPL (lipoprotein lipase) ), VWF (von Villebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transformed growth factor, β1), NPPA (natriuretic peptide precursor A) , IL10 (interleukin 10), EPO (erythropoetin), SOD1 (superoxide dismutase 1, soluble), VCAM1 (vascular cell adhesion molecule 1), IFNG (interferon, γ), LPA (lipoprotein, Lp (a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1), MAPK1 (mitogen-activated protein kinase 1), HP (haptoglobin), F3 (coagulation factor III (thromboplastin, tissue factor)), CST3 (cystatin C), COG2 (Component 2 of oligomeric Gorgi complex), MMP9 (matriuretic metallopeptidase 9 (zelatinase B, 92 kDa zeratinase, 92 kDa IV type collagenase)), SERPINC1 (cellpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (Coagulation Factor VIII, Coagulation Promoting Component), HMOX1 (Hemoxygenase (Decycling) 1), APOC3 (Apolipoprotein C-III), IL8 (Interleukin 8), PROK1 (Prokineticin 1) ), CBS (cystationin β synthase), NOS2 (nitrogen monoxide synthase 2, inducible), TLR4 (Toll-like receptor 4), SELP (SELECTIN P (granular membrane protein 140 kDa, antigen CD62)), ABCA1 (ATP) Binding cassette, subfamily A (ABC1), member 1), AGT (angiotensinogen (selpin peptidase inhibitor, clade A, member 8)), LDLR (low density lipoprotein receptor), GPT (glutamate pyruvate transaminase) Alanine aminotransferase)), VEGFA (vascular endothelial growth factor A), NR3C2 (nuclear receptor subfamily 3, group C, member 2), IL18 (interleukin 18 (interferon-γ inducer)), NOS1 (monooxidation) Nitrogen synthase 1 (nerve type)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrinogen β chain), HGF (hepatocellular growth factor (hepapoetin A;) Scattering factors)), IL1A (interleukin 1, α), RETN (receptor), AKT1 (v-akt mouse thoracic adenoma virus cancer gene homolog 1), LIPC (lipase, liver), HSPD1 (heat shock 60 kDa protein 1 (chaperonin)) )), MAPK14 (mitogen activated protein kinase 14), SPP1 (secretory phosphorus protein 1), ITGB3 (integrin, β3 (platelet glycoprotein 111a, antigen CD61)), CAT (catalase), UTS2 (urotensin 2), THBD ( Thrombomodulin), F10 (coagulation factor X), CP (celluloplasmin (ferroxydase)), TNFRSF11B (tumor necrosis factor receptor superfamily, member 11b), EDNRA (endoserine receptor type A), EGFR (epithelial growth factor receptor) Body (erythroblastic leukemia viral (v-erb-b) cancer gene homologue, bird)), MMP2 (matrix metallopeptidase 2 (zelatinase A, 72 kDa zeratinase, 72 kDa type IV collagenase)), PLG (plasminogen) , NPY (nerve peptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis virus cancer gene homolog (tri)), FN1 (fibronectin) 1), CMA1 (Kimmer Ze 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide-binding protein (G protein), β-polypeptide 3), ADRB2 (adrenalinergic, β-2-, receptor, surface) , APOA5 (apolipoprotein AV), SOD2 (superoxide dismutase 2, mitochondria), F5 (coagulation factor V (proacelelin, unstable factor)), VDC (vitamin D (1,25-dihydroxyvitamin D3)) Receptor), ALOX5 (arachidonate 5-lipoxygenase), HLA-DRB1 (major tissue compatible gene complex, class II, DR β1), PARP1 (poly (ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2), AGER (terminal saccharified product-specific receptor), IRS1 (insulin receptor substrate 1), PTGS1 (prostaglandin endoperoxide synthase 1 (prostaglandin G / H synthase and cyclooxygenase)), ECE1 (endoserine converting enzyme 1), F7 (coagulation factor VII (serum prothrombin conversion promoter)), URN (interleukin 1 receptor antagonist), EPHX2 (epoxidohydrolase 2, cytoplasm), IGFBP1 (insulin-like growth factor binding) Protein 1), MAPK10 (Mightogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily, member 6)), ABCB1 (ATP binding cassette, subfamily B (MDR / TAP), member 1), JUN ( jun cancer gene), IGFBP3 (insulin-like growth factor binding protein 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP-specific), AGTR2 (angiotensin II receptor, type 2), CD40 (CD40 molecule, TNF receptor) Body superfamily member 5), LCAT (lecithin cholesterol acyltransferase), CCR5 (chemokine (CC motif) receptor 5), MMP1 (matrix metallopeptidase 1 (interstitial collagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1) ), ADM (adrenomedurin), DYT10 (dystony 10), STAT3 (signal transduction and transcriptional activator 3 (acute phase receptor)), MMP3 (matrix metallopeptidase 3 (stro) Melaicin 1, Proseratinase)), ELN (Elastin), USF1 (Upstream Transcription Factor 1), CFH (Complementary Factor H), HSPA4 (Heat Shock 70 kDa Protein 4), MMP12 (Matrix Metallopeptidase 12 (Macrophagee Elastase)), MME (membrane metalloendopeptide), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (catepsin B), ANXA5 (anexin A5), ADRB1 (adrenalinergic, β-1-, Receptor), CYBA (cytochrome b-245, α polypeptide), FGA (fibrinogen α chain), GGT1 (γ-glutamyltransferase 1), LIPG (lipase, endothelium), HIF1A (hypoxic inducer 1, α subunit) (Basic helix-loop-helix transcription factor)), CXCR4 (chemokine (C-X-C motif) receptor 4), PROC (protein C (coagulation factor Va and factor VIIIa inactivator)), SCARB1 (Scavenger receptor class B, member 1), CD79A (CD79a molecule, immunoglobulin-related α), PLTP (phospholipid transfer protein), ADD1 (aducine 1 (α)), FGG (fibrinogen γ chain), SAA1 (serum amyloid) A1), KCNH2 (potassium potential open channel, subfamily H (eg-related), member 2), DPP4 (dipeptidyl peptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (sodium diuretic peptide receptor A) / Guanylic acid cyclase A (atrial sodium diuretic peptide receptor A)), VTN (bitronectin), KIAA0101 (KIAA0101), FOS (FBJ mouse osteosarcoma virus cancer gene homologue), TLR2 (trol-like receptor 2), PPIG ( Peptidyl prolyl isomerase G (cyclophyllin G)), IL1R1 (interleukin 1 receptor, type I), AR (androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), SERPINA1 (cellpin) Peptidase inhibitor, Clade A (α-1 antiproteinase, antitrypsin), member 1), MTR (5-methyltetrahydrofolate homocysteine methyltransferase), RBP4 (retinol-binding protein 4, plasma), APOA4 (Apolipoprotein A-IV), CDKN2A (cyclin-dependent kinase inhibitor 2A (inhibits melanoma, p16, CDK4)), FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptor type B) , ITGA2 (integrin, α2 (C)
D49B, VLA-2 receptor α2 subunit)), CABIN1 (calcinulin binding protein 1), SHBG (sex hormone binding globulin), HMGB1 (high mobility group box 1), HSP90B2P (heat shock protein 90 kDa β (Grp94)) , Member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4), GJA1 (gap binding protein, α1, 43 kDa), CAV1 (caveolin 1, caveora protein, 22 kDa), ESR2 ( Estrogen receptor 2 (ER β)), LTA (phosphotoxin α (TNF superfamily, member 1)), GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, sub Family D, Polypeptide 6), NGF (Nerve Growth Factor (β Polypeptide)), SP1 (Sp1 Transcription Factor), TGIF1 (TGFB Inducible Factor Homeobox 1), SRC (v-src sarcoma (Schmidt-Lupin A-) 2 (Schmidt-Ruppin A-2) virus cancer gene homologue (tri)), EGF (epithelial growth factor (β-urogastron)), PIK3CG (phosphoinositide-3-kinase, catalyst, γ-polypeptide), HLA-A ( Major histocompatibility gene complex, class I, A), KCNQ1 (potassium potential open channel, KQT-like subfamily, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (choline) Kinase α), BEST1 (bestrophin 1), APP (amyloid β (A4) precursor protein), CTNNB1 (catenin (cadherin-related protein), β1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombo)) Spondin receptor)), PRKAB1 (protein kinase, AMP activation, β1 non-catalyzed subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7 family, member A1), CX3CR1 (chemocaine (C-X3-C motif)) ) Receptor 1), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferase), HFE (hemochromatosis), IL17A (interleukin 17A), PTEN (phosphatase)・ Protein Insulin homologue), GSTM1 (glutathione S-transferase μ1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1 (coagulation factor XIII, A1 polypeptide), TTR (transsiletin), FABP4 (fatty acid binding protein 4) , Fat cells), PON3 (paraoxonase 3), APOC1 (apolipoprotein CI), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine (5-hydroxytryptamine) Serotonin) receptor 2A), CSF3 (colony stimulator 3 (granulocytes)), CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11, sub) Family B, Polypeptide 2), PTH (parathyroid hormone), CSF2 (colony stimulator 2 (granulocyte macrophages)), KDR (kinase insertion domain receptor (type III receptor tyrosine kinase)), PLA2G2A (phospholipase A2, Group IIA (platelet, lubricant)), B2M (β-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2 family) (Military)), TCF7L2 (transcription factor 7-like 2 (T cell-specific, HMG box)), BDKRB2 (brazikinin receptor B2), NFE2L2 (nuclear factor (erythrocyte-derived 2) -like 2), NOTCH1 (Notch homolog 1) , Translocation-related (Drosophila), UGT1A1 (UDP glucuronosyl transferase 1 family, polypeptide A1), IFNA1 (interferon, α1), PPARD (peroxysome growth factor activating receptor δ), SIRT1 (sirtuin) (Silent mating type information regulation 2 homolog) 1 (S. S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1 (yellow body-forming-releasing hormone)), PAPPA (pregnancy-related plasma protein A, papalicin 1), ARR3 (alestin 3, retinal (X-alestin)), NPPC ( Sodium diuretic peptide precursor C), AHSP (α-hemoglobin stabilizing protein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13), MTOR (mechanical target of rapamycin (serine / threonine kinase)), ITGB2 ( Integrin, β2 (complementary component 3 receptors 3 and 4 subunits), GSTT1 (glutathione S-transferase θ1), IL6ST (interleukin 6 signaling factor (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase) B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocellular nuclear factor 4, α), SLC6A4 (solute transport carrier family 6 (neurotransmitter transporter, cellotonin)) , Member 4), PLA2G6 (phospholipase A2, VI group (cytoplasmic type, calcium independent)), TNFSF11 (tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solute transport carrier family 8 (sodium / calcium exchange)) Body), member 1), F2RL1 (coagulation factor II (thrombin) receptor-like 1), AKR1A1 (ald-ketreductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase 9 family, member A1), BGLAP (bone γ-carboxyglutamic acid (gla) -containing protein), MTTP (microsome triglyceride transfer protein), MTRR (5-methyltetrahydrofolate homocysteine methyltransferase reductase), SULT1A3 (sulfotransferase family, cytoplasmic type, 1A, phenol selection, Members 3), RAGE (renal tumor antigen), C4B (complementary component 4B (Chido blood type), P2RY12 (purine receptor P2Y, G protein conjugate, 12), RNLS (linalase, FAD-dependent amine oxidase) , CREB1 (cAMP response element binding protein 1), POMC (proopiomelanocortin), RAC1 (ras Related C3 botulinum toxin substrate 1 (rho family, low molecular weight GTP binding protein Rac1)), LMNA (Lamine NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, potential opening type, V type, α sub) Unit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), MIF (macrophage migration inhibitor (glycosylation inhibitor)), MMP13 (matrix metallopeptidase 13 (collagenase 3)), TIMP2 (TIMP metallo) Peptidase inhibitor 2), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor type 22) (Lymphocytes)), MYH14 (myosin, heavy chain 14, nonmuscular), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonin deficiency)), SELPLG (selectin P ligand), AOC3 (amineoxidase, copper) Containing 3 (vascular adhesion protein 1)), CTSL1 (catepsin L1), PCNA (proliferating cell nuclear antigen), IGF2 (insulin-like growth factor 2 (somatomedin A)), ITGB1 (integrin, β1 (fibronectin receptor, βpolypeptide,) Antigen CD29 includes MDF2, MSK12)), CAST (carpastatin), CXCL12 (chemokine (CXX motif) ligand 12 (stroma cell-derived factor 1)), IGHE (immunoglobulin heavy chain constant ε), KCNE1 (Potato Potato Open Channel, Isk Related Family, Member 1), TFRC (Transferin Receptor (p90, CD71)), COL1A1 (Collagen, Type I, α1), COL1A2 (Collagen, Type I, α2), IL2RB (Inter) Leukin 2 receptor, β), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoetin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antibacterial peptide) ), PTPN11 (protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solute transport carrier family 2 (promoting glucose transporter), member 1), IL2RA (interroy Kin 2 receptor, α), CCL5 (chemocaine (CC motif) ligand 5), IRF1 (interferon regulator 1), CFLAR (CASP8 and FADD-like apoptosis regulator), CALCA (calcitonin-related polypeptide α), EIF4E (Eukaryotic translation initiation factor 4E), GSTP1 (glutathione S-transferase π1), JAK2 (Yanus kinase 2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (hepalan sulfate proteoglycan 2) , CCL3 (chemokine (CC motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenalinergic) , Β, receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member 2), MMP8 (matrix metallopeptidase 8 (neutrophil colagenase)), NPR2 (sodium diuretic peptide receptor B / guanyl) Acid cyclase B (atrial sodium diuretic peptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS (glutamyl-prolyl-tRNA synthetase), PPARGC1A (peroxysome growth factor activating receptor γ, coactivator 1α), F12 (coagulation factor XII (Hagemann factor)), PECAM1 (platelet / endothelial cell adhesion molecule), CCL4 (chemocaine (CC motif) ligand 4), SERPINA3 (selpin peptidase inhibitor, clade A (α-1 antipeptidease) , Antitrypsin), Member 3), CASR (Calcium Sensing Receptor), GJA5 (Gap-Binding Protein, α5, 40 kDa), FABP2 (Fatigue-Binding Protein 2, Intestine), TTF2 (Transcribing Termination Factor, RNA Peptide II), PROS1 (Protein S (α)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan, β (43 kDa dystrophin-related glycoprotein)), YME1L1 (YME1-like 1 (S. S. cerevisiae)), CAMP (catericidine antibacterial peptide), ZC3H12A (12A containing zinc finger CCCH type), AKR1B1 (ald-ketreductase family 1, member B1 (aldose reductase)), DES (desmin), MMP7 ( Matrix metallopeptidase 7 (matrilysin, uterus)), AHR (aryl hydrocarbon receptor), CSF1 (colony stimulator 1 (macrophages)), HDAC9 (histon deacetylase 9), CTGF (binding tissue growth factor), KCNMA1 (Large Conductance Calcium Activated Potassium Channel, Subfamily M, α Member 1), UGT1A (UDP Glucronosyl Transtransferase 1 Family, Polypeptide A Complex Genesis), PRKCA (Protein Kinase C, α), COMT (Catecol-β) -Methyltransferase), S100B (S100 calcium-binding protein B), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine receptor D4), CAMK2G (calcium / calmodulin-dependent protein) Kinase IIγ), SLC22A2 (solute transport carrier family 22 (organic cation transporter), member 2), CCL11 (chemokine (CC motif) ligand 11), PGF (B321 placenta growth factor), THPO (thrombopoetin), GP6 ( Glycoprotein VI (platelet)), TACR1 (takikinin receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium potential open channel, Shal-related subfamily, member 1) ), LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1 (platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol) Dehydrogenase 1C (class I), γ polypeptide), ALOX12 (arachidonic acid 12-lipoxygenase), AHSG (α-2-HS-sugar protein), BHMT (betaine-homocysteine methyl transferase), GJA4 (gap binding protein, α4) , 37 kDa), SLC25A4 (solute transporter) Body family 25 (mititritic transport carrier; adenine nucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonic acid 5-lipoxygenase activating protein), NUMA1 (nuclear mitotic protein 1), CYP27B1 (Cytochrome P450, Family 27, Subfamily B, Polypeptide 1), CYSLTR2 (Cistinyl leukotriene receptor 2), SOD3 (Superoxide dismutase 3, extracellular), LTC4S (Leukotriene C4 synthase), UCN (Urocortin), GHRL (Grelin / Obestatin prepropeptide), APOC2 (Apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (10 containing Kerhi repeat and BTB (POZ) domains), TNC (Teneisin C), TYMS (thymidylate synthetase), SHCl (SHC (containing 2 domains of Src homology) transforming protein 1), LRP1 (low density lipoprotein receptor-related protein 1), SOCS3 (suppressor 3 for cytokine signaling), ADH1B (alcohol) Dehydrogenase 1B (class I), β-polypeptide), KLK3 (calicrane-related peptidase 3), HSD11B1 (hydroxysteroid (11-β) dehydrogenase 1), VKORC1 (vitamin K epoxy drectinase complex, subunit 1), SERPINB2 (selpin) Peptidase inhibitor, clade B (ovoalbumin), member 2), TNS1 (tensin 1), RNF19A (ring finger protein 19A), EPOR (erythropoetin receptor), ITGAM (integrin, αM (complementary component 3 receptor 3 sub) Unit)), PITX2 (Paired-like homeodomain 2), MAPK7 (Mightogen-activated protein kinase 7), FCGR3A (IgG Fc fragment, low affinity 111a, receptor (CD16a)), LEPR (Leptin receptor), ENG (Endogrin), GPX1 (glutathione peroxidase 1), GOT2 (glutamate oxaloacetate transaminase 2, mitochondria (aspartate aminotransferase 2)), HRH1 (histamine receptor H1), NR112 (nuclear receptor subfamily 1, group I) , Member 2), CRH Tycotropin-releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (potential-dependent anion channel 1), HPSE (heparanase), SFTPD (surfactant protein D), TAP2 (transporter 2, ATP binding cassette) , Subfamily B (MDR / TAP)), RNF123 (ring finger protein 123), PTK2B (PTK2B protein tyrosine kinase 2β), NTRK2 (neurotrophic tyrosine kinase, receptor type 2), IL6R (interleukin 6 receptor) , ACHE (acetylcholine esterase (Yt blood type)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD (P) H dehydrogenase, quinone 1), NR5A1 (Nuclear Receptor Subfamily 5, Group A, Member 1), GJB2 (Gap Binding Protein, β2, 26 kDa), SLC9A1 (Solute Transport Carrier Family 9 (Sodium / Hydrogen Exchanger), Member 1), MAOA (Monoamine Oxide A), PCSK9 (proprotein convertase subtilicin / kexin type 9), FCGR2A (IgG Fc fragment, low affinity IIa, receptor (CD32)), SERPINF1 (selpin peptidase inhibitor, clade F (α-2 anti-plasmin) , Pigment epithelial-derived factor), member 1), EDN3 (endoserine 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest specific 6), SMPD1 (sphingoeline phosphodiesterase 1, acidic lysosome), UCP2 (deconjugated protein 2) (Military, proton carrier)), TFAP2A (transcription factor AP-2α (activated enhancer binding protein 2α)), C4BPA (complementary component 4-binding protein, α), SERPINF2 (selpin peptidase inhibitor, clade F (α-2) Antiplasmin, pigment epithelium-derived factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placenta (Regan isozyme)), CXCR2 (chemokine (CXC motif) receptor 2), SLC39A3 (Solute Transport Carrier Family 39 (Zinc Transporter), Member 3), ABCG2 (ATP Binding Cassette, Subfamily G (WHITE), Member 2), AD A (adenosine deaminase), JAK3 (yanus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), F11 (coagulation factor XI), ATP7A (ATPase, Cu ++ transport, α-polypeptide), CR1 (complementary component (3b / 4b) receptor 1 (Knops blood type)), GFAP (glial fibrous acidic protein), ROCK1 (Rho-related, coiled coil-containing protein kinase) 1), MECP2 (methyl CpG binding protein 2 (let syndrome)), MYLK (myosin light chain kinase), BCHE (butyrylcholine esterase), LIPE (lipase, hormone sensitivity), PRDX5 (peroxyredoxin 5), ADORA1 (adenosin) A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (chemokine (CXC motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, γ2) ), MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CHGA (chromogranin A (parathyroid secretory protein 1)), IAPP (pancreatic islet amyloid polypeptide), RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase / phosphodiesterase 1) , PTHLH (parathyroid hormone-like hormone), NRG1 (neuregulin 1), VEGFC (vascular endothelial growth factor C), ENPEP (glutamylaminopeptidase (aminopeptidase A)), CEBPB (CCAAT / enhancer binding protein (C / EBP)) , Β), NAGLU (N-acetylglucosaminidase, α-), F2RL3 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1), BDKRB1 (bradikinase receptor) B1), ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif, 13), ELANE (elastase, neutrophil expression), ENPP2 (ectonucleotide pyrophosphatase / phosphodiesterase 2), CISH (cytocytosis-induced SH2-containing protein) , GAST (gastrin), MYOC (myocilin, trabecular meshwork-induced kinase response), ATP1A2 (ATPase, Na + / K + transport) , Α2 polypeptide), NF1 (neurofibromin 1), GJB1 (gap binding protein, β1, 32 kDa), MEF2A (muscle cell enhancer factor 2A), VCL (vincurin), BMPR2 (bone-forming protein receptor, type II () Serine / threonine kinase)), TUBB (tubulin, β), CDC42 (cell division cycle 42 (GTP binding protein, 25 kDa)), KRT18 (keratin 18), HSF1 (heat shock transcription factor 1), MYB (v-myb) Myeloblastopathy virus cancer gene homologue (tri)), PRKAA2 (protein kinase, AMP activation, α2 catalytic subunit), ROCK2 (Rho-related, coiled coil-containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein) Related coagulation inhibitors)), PRKG1 (protein kinase, cGMP-dependent, type I), BMP2 (bone-forming protein 2), CTNND1 (catenin (cadherin-related protein), δ1), CTH (cystathionase (cystathionine γ-riase)) , CTSS (catepsin S), VAV2 (vav2 guanine nucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S) -Transtransferase α1), PPIA (peptidyl prolyl isomerase A (cyclophyllin A)), APOH (apolypoprotein H (β-2-glycoprotein I)), S100A8 (S100 calcium-binding protein A8), IL11 (interleukin 11), ALOX15 (arachidonic acid 15-lipoxygenase), FBRN1 (fiburin 1), NR1H3 (nuclear receptor subfamily 1, group H, member 3), SCD (stearoyl-CoA desaturase (Δ-9-desaturase)), GIP (gastric) Inhibitory polypeptide), CHGB (chromogranin B (secret granin 1)), PRKCB (protein kinase C, β), SRD5A1 (steroid-5-alpha-reductase, α-polypeptide 1 (3-oxo-5α-steroid Δ4-) Dehydrogenase α1)), HSD11B2 (hydroxysteroid (11-β) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-α-D-gala) Kusamine: Polypeptide N-Acetylgalactosaminyl transferase 2 (GalNAc-T2)), ANGPTL4 (Angiopoetin-like 4), KCNN4 (Potato intermediate / small conductance calcium activation channel, Subfamily N, Member 4), PIK3C2A (Phonoinocitide-3-kinase, class 2, α-polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major tissue compatible gene) Complex, Class II, DR β 5), BNIP3 (BCL2 / adenovirus E1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4) regulator), S100A12 (S100 calcium binding protein A12), PADI4 (peptidylarginine) Deminase, type IV), HSPA14 (heat shock 70 kDa protein 14), CXCR1 (chemokine (CXC motif) receptor 1), H19 (H19, imprinted maternal expression transcript (non-protein code)), KRTAP19 -3 (Keratin-related protein 19-3), IDDM2 (insulin-dependent intrinsic diabetes 2),
RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, low molecular weight GTP binding protein Rac2)), RYR1 (lianodine receptor 1 (skeleton)), CLOCK (clock homologue (mouse)), NGFR (nerve growth factor receptor () TNFR superfamily, member 16)), DBH (dopamine β-hydroxylase (dopamine β-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, α4), CACNA1C (calcium channel, potential dependent, L-type) , Α1C subunit), PRKAG2 (protein kinase, AMP activation, γ2 non-catalyzed subunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily) 1, Group H, Member 2), TEK (TEK tyrosine kinase, endothelium), VEGFB (vascular endothelial growth factor B), MEF2C (muscle cell enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2), TNFRSF11A (tumor necrosis factor receptor superfamily, member 11a, NFKB activator), HSPA9 (heat shock 70 kDa protein 9 (mortarin)), CYSLTR1 (cystenyl leukotriene receptor 1), MAT1A (methionine adenosyl transferase I, α) , OPRL1 (opiate receptor-like 1), IMPA1 (inositol (myo) -1 (or 4) -monophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (prosome, macro) Pine (macropine) receptor, α type, 6), PSMB8 (proteasome (prosome, macropine) receptor, β type, 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like 1 (chondral sugar protein-) 39)), ALDH1B1 (aldehyde dehydrogenase 1 family, member B1), PARP2 (poly (ADP-ribose) polymerase 2), STAR (steroid-producing acute regulatory protein), LBP (lipopolysaccharide binding protein), ABCC6 (ATP binding cassette, Subfamily C (CFTR / MRP), member 6), RGS2 (regulator of G protein signaling 2, 24 kDa) ), EFNB2 (efrin-B2), GJB6 (gap binding protein, β6, 30 kDa), APOA2 (apolypoprotein A-II), AMPD1 (adenosine monophosphate deaminase 1), DYSF (disferin, limb band type muscle dystrophy 2B (usually) Chromosomal recessive inheritance)), FDFT1 (farnesyl diphosphate farnesyl transferase 1), EDN2 (endoserine 2), CCR6 (chemocaine (CC motif) receptor 6), GJB3 (gap binding protein, β3, 31 kDa), IL1RL1 ( Interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Valdee-Beadle syndrome 4), CELSR2 (cadherin, EGF LAG 7 transmembrane G-type receptor 2 (flamingo homolog,) Drosophila)), F11R (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronoglucosaminidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant protein) 1 Homolog (yeast)), ATF6 (activated transcription factor 6), KHK (ketohexokinase (fructokinase)), SAT1 (spermidine / spermin N1-acetyltransferase 1), GGH (γ-glutamylhydrolase (conjugase, holylpoly) γ-glutamylhydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solute transport carrier family 4, sodium / hydrogen carbonate ion cotransporter, member 4), PDE2A (phosphodiesterase 2A, cGMP stimulant), PDE3B (phosphodiesterase) 3B, cGMP inhibitory), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin β4, X chain), TXNIP (thioredoxin interacting protein), LIMS1 (LIM and aging cell antigen-like domain 1), RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), FOXO1 (forkhead box O1), PNPLA2 (patatin-like phosphorlipase domain-containing 2), TRH (thyrotropin-releasing hormone), GJC1 (gap-binding protein) , Γ1, 45 kDa), SLC17A5 (solute transport carrier protein) Amily 17 (anion / sugar transporter), member 5), FTO (body fat mass and obesity-related), GJD2 (gap-binding protein, δ2, 36 kDa), PSRC1 (proline / serine-rich coiled coil 1), CSP12 (caspase 12 (caspase 12) Gene / pseudogene)), GPBAR1 (G protein-conjugated bile acid receptor 1), PXK (PX domain-containing serine / threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles) homologue 1 (Drosophila (Drosophila) Drosophila))), PBX4 (pre-B cell leukemia homeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1), 15-Sep (15 kDa serenoprotein), CILP2 (chondral middle layer protein 2), TERC (telomerase RNA) Constituents), GGT2 (γ-glutamyltransferase 2), MT-CO1 (mitochrome coxidase I encoded by mitochondria), and UOX (urate oxidase, pseudogene). Any of these sequences can be the target of the CRISPR-Cas system, for example to address mutations.

In a further embodiment, the chromosomal sequences are Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE (apolipoprotein E), ApoB-100 (apolipoprotein B-100), ApoA (apolipoprotein (a)). , ApoA1 (apolipoprotein A1), CBS (cystatione B-synthase), glycoproteins IIb / IIb, MTHRF (5,10-methylenetetrahydrofolate reductase (NADPH), and combinations thereof. In one iteration, the chromosomal sequences involved in cardiovascular disease and the proteins encoded by the chromosomal sequences are targeted by the CRISPR-Cas system from Cacna1C, Sod1, Pten, Ppar (α), ApoE, leptin, and combinations thereof. Can be selected.

Treatment of Liver and Renal Diseases The present invention also contemplates delivering the CRISPR-Cas system described herein, eg, the Cas9 effector protein system, to the kidney. Delivery strategies for inducing cellular uptake of therapeutic nucleic acids include physical force or vector systems such as virus-based, lipid-based or complex-based delivery, or nanocarriers. Wide range of gene therapy to target post-transcriptional events in vivo in various animal renal disease models since early application with low clinical significance expected when nucleic acids are systemically delivered to renal cells by hydrodynamic hyperbaric injection Viral and non-viral carriers have already been applied (Csaba Revezz and Peter Hamar (2011), "Delivery Methods to Target RNAs in the Kidney", Gene Therapy, Gene Therapy. Kang (Ed.), ISBN: 978-953-307-541-9, InTech, available from: http://www.intechopen.com/books/gene-therapy-applications/delivery-methods- -RNAs-inthe-kidney). As a method of delivery to the kidney, in vivo delivery of small interfering RNA (siRNA) targeting the 12 / 15-lipoxygenase (12 / 15-LO) pathway of arachidonic acid metabolism is performed in a streptozotocin-injected type 1 diabetic mouse model. Yuan et al. Investigated whether renal damage and diabetic nephropathy (DN) could be improved. (Am J Physiol Renal Physiol 295: F605-F617, 2008) can be mentioned. To achieve more in vivo arrival and siRNA expression in the kidney, Yuan et al. Used a double-stranded 12 / 15-LO siRNA oligonucleotide conjugated with cholesterol. Approximately 400 μg of siRNA was injected subcutaneously into the mice. Yuang et al. Method can be applied to the CRISPR Cas system of the present invention, intended for subcutaneous injection of 1-2 g of CRISPR Cas conjugated to cholesterol for delivery to the kidney to humans.

Molitoris et al. (JAm Soc Nephrol 20: 1754-1764, 2009) is targeted to p53, the central protein of the apoptotic pathway, utilizing proximal tubular cells (PTCs) as oligonucleotide reabsorption sites in the kidney. The effectiveness of siRNA in preventing renal damage was tested. Naked synthetic siRNA for p53 injected intravenously 4 hours after ischemic injury provided maximum protection for both PTC and renal function. Molitoris et al. Data indicate that rapid delivery of siRNA to proximal tubular cells occurs after intravenous administration. For dose-response analysis, rats were given 0.33; 1, 3, or 5 mg / kg doses of siP53 at the same 4 time points to give cumulative doses of 1.32; 4, 12, and 20 mg / kg, respectively. Was injected into. All siRNA doses tested produced a SCr-lowering effect on day 1, and higher doses were effective for about 5 days compared to PBS-treated ischemic control rats. Cumulative doses of 12 and 20 mg / kg provided the best protective effect. Molitoris et al. Method can be applied to the nucleic acid targeting system of the present invention with the intention of cumulative doses of 12 and 20 mg / kg for delivery to the kidney to humans.

Thompson et al. (Nucleic Acid Therapeutics, Volume 22, Number 4, 2012) reports the toxicity and pharmacokinetic properties of synthetic small interfering RNA I5NP after intravenous administration in rodents and non-human primates. I5NP is designed to temporarily inhibit the expression of the apoptosis-promoting protein p53 by acting through the RNA interference (RNAi) pathway, during acute ischemia / reperfusion injury, such as major cardiac surgery. It has been developed to protect cells from possible acute renal injury and possible post-transplant organ dysfunction after kidney transplantation. A dose of 800 mg / kg I5NP for rodents and 1,000 mg / kg I5NP for non-human primates is required to induce adverse effects, which in monkeys quasi-clinical activation and coagulation time of complement. It has been identified to lead to effects on blood, including a slight increase in. No further adverse effects were observed in rat analogs of I5NP in rats, so these effects contributed to the class action of synthetic RNA duplexes rather than the toxicity associated with the intended pharmacological activity of I5NP. It is shown that it is likely to correspond. Taken together, these data support clinical trials of intravenous administration of I5NP for the preservation of renal function after acute ischemia / reperfusion injury. The NOAEL in monkeys was 500 mg / kg. No effects on cardiovascular, respiratory, and neurological parameters were observed after intravenous administration at dose levels up to 25 mg / kg in monkeys. Therefore, similar dosages can be contemplated for intravenous administration of CRISPR Cas to the human kidney.

Shimazu et al. (JAm Soc Nephrol 21: 622-633, 2010) has developed a system for targeted delivery of siRNA to the glomerulus by a poly (ethylene glycol) -poly (L-lysine) based vehicle. The siRNA / nanocarrier complex was about 10-20 nm in diameter and was sized to allow the complex to pass through the fenestrated endothelium and reach mesangium. After intraperitoneal injection of the fluorescently labeled siRNA / nanocarrier complex, Shimazu et al. Detected siRNA over a long period of time during blood circulation. Repeated intraperitoneal administration of mitogen-activated protein kinase 1 (MAPK1) siRNA / nanocarrier complex suppressed glomerular MAPK1 mRNA and protein expression in a mouse model of glomerulonephritis. Cy5-labeled siRNA complexed with PIC nanocarriers (0.5 ml, 5 nmol siRNA content), naked Cy5-labeled siRNA (0.5 ml, 5 nmol), or Cy5-labeled encapsulated in HVJ-E to examine siRNA accumulation. SiRNA (0.5 ml, 5 nmol content of siRNA) was administered to BALBc mice. Shimazu et al. Method can be applied to the nucleic acid targeting system of the invention, intended for intraperitoneal administration and delivery to the kidney to humans at a dose of about 10-20 μmol CRISPR Cas complexed with a nanocarrier in about 1-2 liters. ..

Treatment of Epithelial and Pulmonary Diseases The present invention also contemplates delivering the CRISPR-Cas system described herein, eg, the Cas9 system, to one or both lungs.

Initially, AAV-2 based vectors were proposed for CFTR delivery to the CF airways, but other serotypes, such as AAV-1, AAV-5, AAV-6, and AAV-9, are genes in various lung epithelial models. It exhibits improved introduction efficiency (see, eg, Li et al., Molecular Epithelium, vol. 17 no. 12, 2067-2077 Dec 2009). AAV-1 has about 100-fold higher transduction efficiency of human airway epithelial cells in vitro than AAV-2 and AAV-55, however, for mouse tracheal airway epithelium, AAV-1 is in vivo and AAV- It was demonstrated that transduction was performed with the same efficiency as 5. In other studies, AAV-5 was 50-fold more efficient in gene delivery to human airway epithelium (HAE) in vitro than AAV-2, and significantly more efficiently in mouse lung airway epithelium in vivo. It is shown. AAV-6 has also been shown to be more efficient in human airway epithelial cells in vitro and in mouse airways in vivo compared to AAV-2 8. A recent isolate, AAV-9, has been shown to exhibit higher gene transfer efficiencies than AAV-5 in mouse nasal and alveolar epithelium in vivo, and gene expression was detected over a 9-month period, indicating that the CFTR gene It is suggested that long-term gene expression in vivo, which is a desirable property for delivery vectors, can be achieved with AAV. Furthermore, it has been demonstrated that AAV-9 can be re-administered to mouse lung without loss of CFTR expression and with minimal immunological consequences. The apical surface of CF and non-CF HAE cultures can be inoculated with 100 μl of AAV vector for several hours (see, eg, Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009). MOI can vary from 1 × 10 ³ to 4 × 10 ⁵ vector genome / cell depending on virus concentration and experimental purpose. The vectors cited above are intended for delivery and / or administration of the present invention.

Zamora et al. (Am J Respir Crit Care Med Vol 183.pp 531-538, 2011) is an example of application of an RNA interference therapeutic agent for the treatment of human infections, and a lung transplantation recipe infected with respiratory syncytial virus (RSV). We reported a randomized trial of antiviral drugs in Ent. Zamora et al. Conducted a randomized, double-blind, placebo-controlled trial in LTX recipients of RSV respiratory tract infections. Patients were allowed to receive standard treatment for RSV. Aerosolized ALN-RSV01 (0.6 mg / kg) or placebo was administered daily for 3 days. This study demonstrates that RNAi therapeutics that target RSV can be safely administered to LTX recipients with RSV infection. Three daily doses of ALN-RSV01 did not result in exacerbation of airway symptoms or pulmonary dysfunction and did not exhibit systemic inflammatory effects such as induction of cytokines or CRP. Pharmacokinetics showed a slight low transient systemic exposure after inhalation, which was due to ALN-RSV01 administered intravenously or by inhalation from the circulation by exonuclease-mediated digestion and renal excretion. Consistent with preclinical animal data showing rapid disappearance. Zamora et al. The method can be applied to the nucleic acid targeting system of the present invention, wherein aerosolized CRISPR Cas can be contemplated in the present invention, for example, at a dosage of 0.6 mg / kg.

Schwank et al. (Cell Stem Cell, 13: 653-58, 2013) used CRISPR-Cas9 to repair defects associated with cystic fibrosis in human stem cells. The target of this team was the gene for ion channels, the cystic fibrosis transmembrane conductor receptor (CFTR). Deletions in CFTR cause false protein folding in patients with cystic fibrosis. Schwank et al. Can correct this defect using CRISPR with a donor plasmid containing the repair sequence to be inserted using cultured intestinal stem cells generated from cell samples from two cystic fibrosis children. did it. The researchers then grew cells into intestinal "organoids," or small intestines, and showed that they functioned normally. In this example, about half of the cloned organoids had appropriate genetic modification.

Treatment of Muscular Diseases The present invention also contemplates delivering the CRISPR-Cas systems described herein to muscles, such as the Cas9 system.

Bortolanza et al. (Molecular Therapy vol. 19 no. 11, 2055-264 Nov. 2011) showed that systemic delivery of RNA interference expression cassettes in FRG1 mice after the development of facioscapulohumeral muscular dystrophy (FSHD) was performed without signs of toxicity. It has been shown to result in dose-dependent long-term FRG1 knockdown. Bortolanza et al. Found that a single intravenous injection of 5 × 10 ¹² vg of rAAV6-sh1FRG1 rescues muscle histopathology and muscle function in FRG1 mice. Specifically, 200 μl containing a 2 × 10 ¹² or 5 × 10 ¹² vg vector in physiological solution was injected into the tail vein using a 25 gauge Terumo syringe. Bortolanza et al. The method of is applied to AAV expressing CRISPR Cas and can be injected into humans at a vector dosage of about 2 × 10 ¹⁵ or 2 × 10 ¹⁶ vg.

Dumonseaux et al. (Molecular Therapy vol.18 no. 5,881-887 May 2010) inhibits the myostatin pathway using the technique of RNA interference (sh-AcvRIIb) on the myostatin receptor AcvRIIb mRNA. The vectorized U7 exon skipping technique (U7-DYS) mediated the recovery of pseudodystrophin. An adeno-associated vector carrying either the sh-AcvrIIb construct alone, the U7-DYS construct alone, or a combination of both constructs was injected into the tibialis anterior (TA) muscle of dystrophy mdx mice. Injection was performed with ^{10 11} AAV viral genome. Dumonseaux et al. Method can be applied to AAV expressing CRISPR Cas and injected into humans, for example, at a vector dosage of about 10 ¹⁴ to about 10 ¹⁵ vg.

Kinouchi et al. (Gene Therapy (2008) 15, 1126-1130) is effective in delivering in vivo siRNA to skeletal muscle of normal or affected mice using nanoparticulate formulations of unmodified siRNA containing atelocollagen (ATCOL). Report sex. Topical application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, into mouse skeletal muscle or veins via ATCOL mediation resulted in a marked increase in muscle mass within weeks of administration. These results imply that ATCOL-mediated application of siRNA is a powerful tool for further therapeutic applications in diseases including muscular atrophy. MstsiRNA (final concentration 10 mM) was mixed with ATCOL (final concentration 0.5% for topical administration) (AteloGene, Koken, Tokyo, Japan) according to the manufacturer's instructions. After anesthesia of mice (20-week-old male C57BL / 6) with Nembutal (25 mg / kg, ip), the Mst-siRNA / ATCOL complex was injected into the masticatory and biceps femoris muscles. Kinouchi et al. The method of is applied to CRISPR Cas and can be injected into humans muscle, for example with a dosage of about 500-1000 ml of a 40 μM solution. Hagström et al. (Molecular Therapy Vol. 10, No. 2, August 2004) describes an intravascular non-viral method that enables efficient and repeatable nucleic acid delivery to muscle cells (muscle fibers) throughout mammalian limb muscles. ing. This procedure involves injection of naked plasmid DNA or siRNA into the distal vein of the extremity that has been transiently blocked with a tourniquet or blood pressure cuff. Nucleic acid delivery to muscle fibers is facilitated by rapid injection in a volume sufficient to allow the nucleic acid solution to overflow into the muscle tissue. High transgene expression in skeletal muscle was achieved with minimal toxicity in both small and large animals. Evidence of siRNA delivery to limb muscles was also obtained. For intravenous injection of plasmid DNA into rhesus monkeys, a three-way stopcock was connected to two syringe pumps (Model PHD 2000; Hard Instruments), each loaded with a single syringe. Five minutes after papaverine injection, pDNA (15.5 to 25.7 mg in 40-100 ml saline) was injected at a rate of 1.7 or 2.0 ml / sec. This can be scaled up for plasmid DNA expressing the CRISPR Cas of the present invention to an injection of about 300-500 mg in 800-2000 ml saline for humans. The adenoviral vector injection to rat, Normal saline 3 ml (NSS) in 2 × 10 ⁹ pieces of infectious particles were injected. This can be scaled up for the adenovirus vector expressing CRISPR Cas of the invention to inject about 1 × 10 ¹³ infectious particles in 10 liters of NSS for humans. For siRNA, rats were injected with 12.5 μg of siRNA into the great saphenous vein and primates were injected with 750 μg of siRNA into the great saphenous vein. This can be scaled up for the CRISPR Cas of the present invention, eg, an injection of about 15 to about 50 mg into the great saphenous vein of a human.

For example, see also International Publication No. 2013163628 A2 pamphlet, "Genetic Direction of Mutated Genes," which is a public application of the University of Duke (Duke University), for example, suddenly of a dystrophin gene. Immature termination that can be repaired with nuclease-mediated heterologous end binding, including those involved in Duchenne muscular dystrophy (“DMD”), a recessive, lethal X-linked disorder that causes muscle degeneration due to mutations. It describes an attempt to repair frame-shifted mutations that give rise to codon and truncated gene products. The majority of dystrophin mutations that cause DMD are exon deletions, which disrupt the reading frame and cause premature translation termination of the dystrophin gene. Dystrophin is a cytoplasmic protein that provides structural stability to the dystroglycan complex of the cell membrane, which is involved in the regulation of muscle cell integrity and function. The dystrophin gene or "DMD gene" as used synonymously herein is on a 2.2 megabase and is located at locus Xp21. The primary transcription is about 2,400 kb and the mature mRNA is about 14 kb. 79 exons encode the protein, which exceeds 3500 amino acids. Exon 51 is often flanked by frame-destroying deletions in DMD patients, which has been targeted in clinical trials for oligonucleotide-based exon skipping. A clinical trial of the exon 51 skipping compound eteprilsen recently reported significant functional benefits over 48 weeks, averaging 47% of dystrophin-positive fibers compared to baseline. Mutations in exon 51 are ideally suitable for permanent repair by NHEJ-based genome editing.

The method of U.S. Patent Application Publication No. 20130145487, transferred to Cellectis, which is a method for a meganuclease variant that cleaves a target sequence from a human dystrophin gene (DMD), has also been modified for the nucleic acid targeting system of the invention. can do.

Treatment of Skin Diseases The present invention also contemplates delivering the CRISPR-Cas systems described herein to the skin, such as the Cas9 effector protein system.

Hickerson et al. (Molecular Therapy-Nucleic Acids (2013) 2, e129) relates to an electric microneedle array skin delivery device for delivering self-delivery (sd) siRNA to human and mouse skin. A major challenge in the clinical transition of siRNA-based dermatological agents is the development of effective delivery systems. Many attempts have been made to various skin delivery techniques, but their success is limited. In clinical trials where the skin was treated with siRNA, the severe pain associated with subcutaneous needle injection prevented further patient enrollment in the trial, resulting in an improved, more "patient-friendly" (ie, almost painless or less). The need for a delivery method (not at all) is highlighted. Microneedles are an efficient way to deliver large charged cargoes containing siRNA over the stratum corneum, which is the largest barrier, and are generally considered to be less painful than conventional subcutaneous needles. The electric "stamp type" microneedle device is available from Hickerson et al. Hairless mouse tests have shown safety, including the electric microneedle array (MMNA) device used by, and (i) extensive use in the cosmetics industry, and (ii) nearly limited testing. Subcutaneous delivery of siRNA using this device causes little or no pain, as evidenced by all volunteers admitting that the use of this device is much less painful than vaccination against influenza. It is suggested that the pain is much less than that experienced in previous clinical trials using needle injection. The MMNA device (commercially available as Triple-M or Tri-M from Bomtech Electrical Co, Seoul, South Korea) was configured for delivery of siRNA to mouse and human skin. An sd-siRNA solution (up to 300 μl of 0.1 mg / ml RNA) was introduced into the chamber of a disposable Tri-M needle cartridge (Bomtech) set to a depth of 0.1 mm. For the treatment of human skin, unspecified skin (obtained immediately after the surgical procedure) was manually stretched and pinned to a cork platform prior to the procedure. All intradermal injections were performed using an insulin syringe equipped with a 28 gauge 0.5 inch needle. Hickerson et al. MMNA devices and methods can be used and / or adapted for the delivery of CRISPR Cas of the invention, eg, at doses of up to 300 μl 0.1 mg / ml CRISPR Cas to the skin.

Leachman et al. (Molecular Therapy, vol.18 no.2,442-446 Feb.2010) is a life-threatening sole using a first small interfering RNA (siRNA) -based skin treatment. It relates to a phase Ib clinical trial for the treatment of congenital nail hyperplasia (PC), a rare skin disorder that is an autosomal dominant syndrome including keratopathy. This siRNA, called TD101, specifically and strongly targets keratin 6a (K6a) N171K mutant mRNA without affecting wild-type K6a mRNA.

Zheng et al. (PNAS, July 24, 2012, vol.109, no.30, 11975-11980) are spherical nucleic acid nanoparticles in which the gold core is surrounded by a dense shell of highly oriented covalently immobilized siRNA. It has been shown that conjugates (SNA-NC) are free to pass through almost 100% of in vitro keratinocytes, mouse skin, and human epidermis within hours of application. Zheng et al. Has demonstrated that a single application of 25 nM epidermal growth factor receptor (EGFR) SNA-NC over 60 hours demonstrates effective gene knockdown in human skin. Similar dosages are contemplated for CRISPR Cas immobilized on SNA-NC for administration to the skin.

Overview of Gene Therapy Examples of disease-related genes and polynucleotides that can be targeted in the practice of the present invention and specific information on the disease are available on the worldwide web at the Johns Hopkins University Institute of Genetic Medicine (McKusick). -Naths Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) And National Center for Biotechnology Information (National Center for Biotechnology, Organization, National Center for Biotechnology) it can.

Mutations in these genes and pathways can result in the production of inappropriate proteins or inappropriate amounts of proteins that affect function. Further examples of genes, diseases and proteins are incorporated by reference herein from US Provisional Patent Application No. 61 / 736,527 filed December 12, 2012. Such genes, proteins and pathways can be target polynucleotides for the CRISPR complex of the invention.

Embodiments of the invention also relate to methods and compositions relating to gene knockout, gene amplification and repair of specific mutations associated with DNA repeat instability and neurological disorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Institutes and Neurological Diseases, Second Edition, Academic Press, Oct 13, 2011-Medical). Specific aspects of tandem repeat sequences have been shown to be involved in more than 20 human diseases ("New insights into repeat instability: roll of RNA / DNA hybrids". ) ”. McIvor EI, Polak U, Napierala M. RNA Biol. 2010 Sep-Oct; 7 (5): 551-8). This effector protein system can be used to correct these defects in genome instability.

Some further aspects of the invention are further described in the topic bar Genetic Disorders (website at health.nih.gov/topic/GeneticDisorders) on the website of the National Institutes of Health. Regarding the correction of defects associated with a wide range of genetic disorders. Genetic brain diseases include, but are not limited to, adrenal leukodystrophy, thyroid deficiency, Icardi syndrome, Alpers disease, Alzheimer's disease, Bath syndrome, Batten's disease, CADASIL, cerebral degeneration, Fabry's disease, Gerstmann- Stroisler-Shineker's disease, Huntington's disease and other triplet repeat diseases, Lee's disease, Resh-Naihan's syndrome, Menkes' disease, mitochondrial myopathy and NINDS colpothefary can be mentioned. These diseases are further described in the bar Genetic Brain Disorders on the National Institutes of Health website.

Illustrative Use of CRISPR Cas Systems The present invention encodes non-naturally occurring or engineered compositions, or components of said compositions, used to modify target cells in vivo, ex vivo or in vitro. An expression in which a vector or delivery system containing one or more polynucleotides or one or more polynucleotides encoding a component of the composition is provided, and the progeny or cell line of the cell modified by CRISPR is changed after the modification. It can be done by altering the cells to retain their mold. The modified cells and progeny can be part of a multicellular organism such as a plant or animal in which the CRISPR system has been applied ex vivo or in vivo to the desired cell type. The CRISPR invention can be a therapeutic method. Therapeutic treatment methods may include gene or genome editing, or gene therapy.

Modification of Target by CRISPR-Cas System or Complex In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal, and modifying one or more cells. Culturing can be done in Exobibo at any stage. The one or more cells may be further reintroduced into a non-human animal or plant. For cells to be reintroduced, it is particularly preferred that the cells are stem cells.

In some embodiments, the method comprises attaching the CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex is: Contains a CRISPR enzyme that hybridizes to or hybridizes to a target sequence within the target polynucleotide, the guide sequence is linked to a tracr mate sequence, and then the tracr mate sequence It hybridizes to the tracr sequence.

In one aspect, the invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises the step of binding a CRISPR complex to a polynucleotide that causes an increase or decrease in expression of the polynucleotide; where the CRISPR complex is said to be the poly. It contains a CRISPR enzyme that hybridizes to a target sequence within a nucleotide or forms a complex with a guide sequence capable of hybridizing to the guide sequence, the guide sequence being linked to a tracr mate sequence, and then the tracr mate sequence hybridizing to the tracr sequence. To soy. Similar considerations and conditions apply to methods of modifying the target polynucleotide as described above. In practice, these sampling, culturing and reintroduction options apply to all aspects of the invention.

In fact, in any aspect of the invention, the CRISPR complex can include a CRISPR enzyme that hybridizes to or forms a complex with a guide sequence capable of hybridizing to the target sequence, wherein the guide sequence is a tracr mate sequence. The tracr mate sequence may then hybridize to the tracr sequence.

Similar considerations and conditions apply to methods of modifying the target polynucleotide as described above. Thus, it comprises at least one modification of any of the non-naturally occurring CRISPR enzymes described herein, whereby the enzyme has certain enhanced abilities. In particular, any of these enzymes is capable of forming a CRISPR complex with a guide RNA. During the formation of such a complex, the guide RNA is capable of binding to the target polynucleotide sequence and the enzyme is capable of modifying the target locus. In addition, the enzymes in the CRISPR complex have a reduced ability to modify one or more off-target loci when compared to unmodified enzymes.

In addition, the modified CRISPR enzymes (emzyme) described herein include enzymes that have an increased ability to modify one or more target loci when compared to non-modified enzymes in the CRISPR complex. Such a function may be provided separately from, or in combination with, the above-mentioned function of reducing the ability to modify one or more off-target loci. The CRISPR enzyme as described herein, such as any such enzyme in combination with any activity provided by one or more associated heterologous functional domains, any further mutation that reduces nuclease activity, etc. May be provided with any of the further modifications to.

In an advantageous embodiment of the invention, the ability to modify one or more off-target loci when compared to an unmodified enzyme and the ability to modify one or more target loci when compared to an unmodified enzyme. A modified CRISPR enzyme (emzyme) with an increase is provided. In combination with further modifications to the enzyme, a significant increase in specificity can be achieved. For example, a combination of such an advantageous embodiment and one or more additional mutations is provided, where the one or more additional mutations are in one or more catalytically active domains. Such additional catalytic mutations may confer nickase function as described in detail elsewhere herein. In such an enzyme, the specificity is improved in terms of enzyme activity, so that the specificity can be enhanced.

Modifications that reduce the off-target effect and / or enhance the on-target effect as described above are performed on amino acid residues located in the positively charged region / groove between the RuvC-III domain and the HNH domain. Can be It is understood that any of the functional effects described above can be achieved by modifying the amino acids in the groove as described above, but can also be achieved by modifying the amino acids adjacent to or outside the groove. Will be done.

Further features that can be engineered to result in a modified CRISPR enzyme as described herein include: 1. 1. A modified CRISPR enzyme that disrupts DNA: protein interactions without affecting protein tertiary or secondary structure. This includes residues that make contact with any part of the RNA: DNA double strand. 2. 2. A modified CRISPR enzyme that weakens the protein-protein interactions that Cas9 carries to take the conformation essential for nuclease cleavage in response to DNA binding (on or off-target). For example: A modification that slightly inhibits the nuclease conformation of the HNH domain (located in fragile phosphate), but still allows it. 3. 3. A modified CRISPR enzyme that enhances protein-protein interactions that hold Cas9 in a conformation that inhibits nuclease activity in response to DNA binding (on or off-target). For example: A modification that stabilizes the HNH domain in a conformation that keeps it away from the fragile phosphate. Any such additional enhancement may be provided in combination with any other modification to the CRISPR enzyme as described in detail elsewhere herein.

Any of the enhanced functions described herein can be added to any CRISPR enzyme, such as the Cas9 enzyme. The Cas9 enzymes described herein are derived from the Cas9 enzymes of Streptococcus pyogenes (S. pyogenes) and Staphylococcus aureus (S. aureus). However, it will be appreciated that any of the functions described herein can be engineered to Cas9 enzymes from other orthologs, including chimeric enzymes containing fragments from multiple orthologs.

Nucleic acids, amino acids, proteins, regulatory sequences, vectors, etc. The present invention uses nucleic acids that bind to the target DNA sequence. This is advantageous because nucleic acids are much easier and cheaper to make than proteins, and specificity can vary depending on the length of extension for which homology is required. For example, it is not necessary to perform complicated 3D positioning of a plurality of fingers. The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used interchangeably. These terms refer to any length of deoxyribonucleotide or ribonucleotide, or a polymeric form of a nucleotide thereof. The polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) determined by linkage analysis, exons, introns, Messenger RNA (mRNA), transfer RNA, ribosome RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, Isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. The term also includes nucleic acid-like structures with synthetic backbones. For example, Eckstein, 1991; Baserga et al. , 1992; Milligan, 1993; International Publication No. 97/03211 Pamphlet; No. 96/39154 Pamphlet; Mata, 1997; Stratus-Saturday, 1997; and Samstag, 1996. Polynucleotides can include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be done before or after the construction of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization of the nucleotides, for example by joining with labeled components. As used herein, the term "wild-type" is a term understood by those of skill in the art and is distinct from mutant or mutant forms of organisms, strains, in typical naturally occurring forms. It means a gene or characteristic. The "wild type" can be the baseline. The term "mutant" as used herein should be construed to mean the presentation of qualities with patterns that deviate from those naturally occurring. The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate human intervention. When the term refers to a nucleic acid molecule or polypeptide, that the nucleic acid molecule or polypeptide is naturally naturally bound and at least substantially free from at least one other component found in nature. Means. "Complementarity" is the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence by conventional Watson-Crick base pairing or other non-conventional types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5 or 6 out of 10). 7, 8, 9 and 10 are 50%, 60%, 70%, 80%, 90% and 100% complementarity, respectively). By "fully complementary" is meant that all contiguous residues of a nucleic acid sequence are hydrogen bonded to the same number of contiguous residues of a second nucleic acid sequence. The term "substantially complementary" as used herein refers to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97 relative to regions of 24, 25, 30, 35, 40, 45, 50, or more nucleotides. %, 98%, 99%, or 100% refers to the degree of complementarity, or refers to two nucleic acids that hybridize under stringent conditions. As used herein, the "stringent condition" of hybridization is that a nucleic acid having complementarity to a target sequence preferentially hybridizes to the target sequence and is substantially different from a non-target sequence. It is a condition that does not hybridize. Stringent conditions are generally sequence-dependent and depend on the number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions, Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay" , Elsevier, N. et al. Y. It is detailed in. When referring to polynucleotide sequences, complementary or partially complementary sequences are also envisioned. They can preferably hybridize to the reference sequence under highly stringent conditions. Generally, relatively low stringent hybridization conditions are selected to maximize hybridization rates: about 20-25 ° C., which is below the thermal melting point ( _Tm ). This _Tm is the temperature at which 50% of a particular target sequence hybridizes to a fully complementary probe in solution at defined ionic strength and pH. In general, because it requires at least about 85% nucleotide complementarity of hybridised sequences, highly stringent washing conditions are selected to be less about 5 to 15 ° C. than T _m. Since it requires at least about 70% nucleotide complementarity of the hybridized sequence, medium stringent wash conditions are chosen to be about 15-30 ° C, below T _m . Highly acceptable (very low stringent) wash conditions can be 50 ° C. below _Tm , allowing high levels of mismatch between hybridized sequences. For those of skill in the art, other physical and chemical parameters during the hybridization and washing steps will also result in detectable hybridization signals from a particular level of homology between the target and probe sequences. You will find that it can be modified to affect it. Preferred high stringent conditions are incubation at 42 ° C. in 50% formamide, 5 × SSC and 1% SDS, or incubation at 65 ° C. in 5 × SSC and 1% SDS, and 0.2 × SSC. And 65 ° C. washing with 0.1% SDS. "Hybridization" is a reaction in which one or more polynucleotides form a stable complex by hydrogen bonding between the bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogsteen binding, or other sequence-specific methods. The complex may include two strands forming a double chain structure, three or more strands forming a multichain complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction can constitute one step in a broader process, eg, initiation of PCR, or enzymatic cleavage of a polynucleotide. A sequence that can hybridize to a given sequence is called a "complement" of the given sequence. As used herein, the term "genome locus" or "locus" (including the plural) is a particular location of a gene or DNA sequence on a chromosome. A "gene" is an RNA strand that extends a DNA or RNA encoding a polypeptide, or that plays a functional role in an organism and is therefore inherited on a molecular basis in the organism. For the purposes of the present invention, a gene can be considered to contain such regulatory sequences, whether or not the regions that regulate the production of the gene product are flanked by coding and / or transcriptional sequences. .. Thus, genes are, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites, etc. And the locus regulatory region. As used herein, "genome locus expression" or "gene expression" is the process by which information from a gene is used to synthesize a functional gene product. The product of gene expression is often a protein, but in the case of non-protein coding genes such as rRNA or tRNA genes, the product is a functional RNA. The process of gene expression is used by all known life-eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), and viruses that produce viable functional products. "Expression" of a gene or nucleic acid as used herein includes not only gene expression in cells, but also transcription and translation of nucleic acids in cloning systems and other associations. As used herein, "expression" also refers to the process by which a polynucleotide is transcribed from a DNA template (eg, into mRNA or other RNA transcript), and / or the transcribed mRNA followed by a peptide, polypeptide. , Or the process of being translated into protein. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells. The terms "polypeptide,""peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also includes modifications; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other treatments, such as amino acid polymers that have been conjugated to a labeling component. As used herein, the term "amino acid" includes naturally occurring and / or non-naturally occurring or synthetic amino acids, including both glycine and D or L optical isomers, amino acid analogs, and peptide mimetics. As used herein, the term "domain" or "protein domain" refers to a portion of a protein sequence that can exist and function independently of the rest of the protein chain. As described in aspects of the invention, sequence identity is associated with sequence homology. Homology comparisons can be made with the naked eye, more generally with the help of readily available sequence comparison programs. These commercially available computer programs can calculate percent (%) homology between two or more sequences, and can also calculate sequence identity shared by two or more amino acid or nucleic acid sequences. it can.

In aspects of the invention, the term "guide RNA" refers to a polynucleotide sequence comprising one or more of a putative or identified tracr sequence and a putative or identified crRNA or guide sequence. In a detailed embodiment, the "guide RNA" comprises a putative or identified crRNA sequence or guide sequence. In a further embodiment, the guide RNA does not contain a putative or identified tracr sequence.

As used herein, the term "wild type" is a term of the art understood by those skilled in the art, as naturally occurring as distinct from mutants or mutant forms. Means a typical form of an organism, strain, gene, or characteristic of. The "wild type" can be the baseline.

As used herein, the term "mutant" shall be construed to mean the presentation of a quality with a pattern that deviates from what is naturally occurring.

The terms "non-naturally occurring" or "engineered" are used synonymously to indicate human intervention. When the term refers to a nucleic acid molecule or polypeptide, the nucleic acid molecule or polypeptide is at least one other component that is naturally associated with it in nature and as found in nature. It means that it is practically not included. It is understood that in all embodiments and embodiments, whether or not these terms are included, preferably the may be optional and therefore preferably included or not included. There will be. In addition, the terms "non-naturally occurring" and "engineered" may be used synonymously and therefore can be used alone or in combination, replacing either one with a combined description. May be good. In particular, "engineered" is preferred instead of "non-naturally occurring" or "non-naturally occurring and / or engineered".

Sequence homology can be determined by any of a number of computer programs known in the art, such as BLAST or FASTA. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit Package (University of Wisconsin, USA; Deverex et al., 1984, Nucleic Acids Research 12: 387). Examples of other software capable of performing sequence comparisons include, but are not limited to, BLAST packages (see Ausubel et al., 1999 supra-Chapter 18), FASTA (Atschul et al., 1990, J. Mol). Biol., 403-410), and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online search (see Ausubel et al., 1999 supra, pp. 7-58-7-60). However, it is preferable to use the GCG Bestfit program. Percentage (%) sequence homology may be calculated for contiguous sequences, i.e., aligning one sequence with the other, each amino acid or nucleotide in one sequence with the corresponding amino acid or nucleotide in the other sequence. Each residue is directly compared. This is called a "no gap" alignment. Such gapless alignment is performed on a relatively small number of residues. This is a very simple and consistent method, but cannot take into account, for example, that subsequent amino acid residues are out of alignment due to one insertion or deletion in the same sequence pair. Therefore, global alignment can potentially significantly reduce% homology. As a result, most sequence comparison methods are designed to generate optimal alignments that take into account possible insertions or deletions without undue penalties for the overall homology or identity score. To. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology or identity. However, these more complex methods assign a "gap penalty" to each gap that appears in the alignment, so for the same number of the same amino acids, a sequence alignment with as few gaps as possible (between two comparable sequences). (Reflecting high relevance) can achieve higher scores than sequences with many gaps. Typically, "Affinity gap costs" are used that impose a relatively high cost on the existence of a gap and reduce the penalty for each subsequent residue in that gap. This is the most commonly used gap scoring system. High gap penalties can, of course, produce optimized alignments with smaller gaps. In many alignment programs, it is possible to change the gap penalty. However, when using software for sequence comparison, it is preferable to use the default value. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for the gap and -4 for each extension. Therefore, in order to calculate the maximum% homology, it is necessary to first consider the gap penalty to generate the optimum alignment. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (Deverex et al., 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, BLAST package (Ausubel et al., See ^{1999 Short Protocols in Molecular Biology, 4} th Ed.-Chapter 18), FASTA (Altschul Et al., 1990 J. Mol. Biol. 403-410), and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online search (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pp. 7-58-7-60). However, for some applications it is preferred to use the GCG Bestfit program. A novel tool called BLAST 2 Technologies is also available for comparison of protein and nucleotide sequences (FEMS Microbiol Lett. 1999 174 (2): 247-50; FEMS Microbiol Lett. 1999 177 (1): 187-8 and See the website of the National Center for Biotechnology Information on the website of the National Institutes for Health (see the website of the National Center for Biotechnology Information). The final% homology can be measured in terms of identity, but the alignment process itself is not typically based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLASTUM62 matrix-the default matrix of the BLAST program suite. The GCG Wisconsin program generally uses either the official default values or, if available, a custom symbol comparison table (see the user manual for further details). For some applications, it is preferable to use the official default values for GCG packages, or for other software, use default matrices such as BLOSUM62. Alternatively, the percentage homology is calculated using the multiple alignment function of DNASIS ™ (Hitachi Software) based on the same algorithm as Clustal (Higgins DG & Sharp PM (1988), Gene 73 (1), 237-244). You may. Once the software has generated the optimal alignment, it is possible to calculate% homology, preferably% sequence identity. Software typically does this as part of a sequence comparison and produces numerical results. The sequence can also have deletions, insertions or substitutions of amino acid residues that result in silent changes resulting in functionally equivalent substances. Planned amino acid substitutions may be made based on similarities in amino acid properties (such as residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties), thus making amino acids functional. It is useful to put them together in various groups. Amino acids may be grouped based solely on the properties of their side chains. However, it is even more useful to include mutation data as well. The set of amino acids thus obtained appears to be conserved for structural reasons. These pairs can be described in the form of Venn diagrams (Livingstone CD and Barton GJ (1993) "Protein sequence alignment: a strategy for the hierarchical". analog. -218). Conservative substitutions can be made, for example, according to the table below, which describes the generally accepted Venn diagram amino acid classification.

The terms "subject", "individual", and "patient" are used herein synonymously to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murids, primates, humans, agricultural animals, athletic animals, and pets. Also included are tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.

The terms "therapeutic agent", "therapeutic agent" or "therapeutic agent" are used synonymously to refer to a molecule or compound that imparts some beneficial effect when administered to a subject. Beneficial effects include feasibility of diagnostic judgment; improvement of disease, symptom, disorder, or pathological condition; reduction or prevention of the onset of disease, symptom, disorder or condition; and disease, symptom, disorder or disease. Includes general counter-conditions.

As used herein, "treat" or "treat", or "alleviate" or "improve" are used synonymously. These terms refer to methods for achieving beneficial or desired outcomes, including but not limited to therapeutic and / or prophylactic benefits. Therapeutic benefit means any therapeutically relevant improvement or effect on one or more diseases, conditions, or conditions under treatment. For prophylactic benefits, the composition is a subject at risk of developing a particular disease, condition, or condition, or the physiological condition of the disease, even if the disease, condition, or condition may not yet be present. Can be administered to a subject complaining of one or more of.

The term "effective amount" or "therapeutically effective amount" refers to an amount of drug sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the subject and disease condition under treatment, the weight and age of the subject, the severity of the disease condition, the administration method, etc., which can be easily determined by those skilled in the art. be able to. The term also applies to doses that may provide images for detection by any one of the imaging methods described herein. Specific doses are among the finely selected agents, dosing regimens to be followed, whether administered in combination with other compounds, timing of administration, tissue to be imaged, and the physical delivery system on which it is supported. It can vary depending on one or more.

Unless otherwise indicated, the practice of the present invention utilizes prior arts of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA within the scope of the art in the art. .. Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRET PROTOCOLS IN MOLECULAR BIOLOGY (F.M.A.Ess) , Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

Some aspects of the invention relate to a vector system comprising one or more vectors, or the vector itself. Vectors can be designed for expression of CRISPR transcripts (eg, nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, the CRISPR transcript can be expressed in bacterial cells, such as Escherichia coli, insect cells (using a baculovirus expression vector), yeast cells, or mammalian cells. For suitable host cells, see Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro using, for example, the T7 promoter regulatory sequence and T7 polymerase.

Embodiments of the invention are sequences that may include homologous substitutions, where both substitution and exchange are used to refer to the replacement of an existing amino acid residue or nucleotide with another amino acid residue or nucleotide. Containing (both polynucleotides or polypeptides), this homologous substitution, ie, in the case of amino acids, is a homologous substitution, eg, a basic basic substitution, an acidic acidic substitution, a polar polarity substitution. Etc. can occur. Non-homologous substitutions, that is, substitutions from one class of residues to another, or non-naturally occurring amino acids such as ornithine (hereinafter referred to as Z), ornithine diaminobutyrate (hereinafter referred to as B). , Norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine, and substitutions involving the uptake of phenylglycine can also occur. The mutant amino acid sequence may contain a suitable spacer group between any two amino acid residues of the sequence, which may include an amino acid spacer, such as a glycine or β-alanine residue, as well as an alkyl group, such as an alkyl group. , Methyl group, ethyl group, or propyl group. Further forms of mutation with the presence of one or more amino acid residues in the peptoid form will be well understood by those of skill in the art. For the avoidance of doubt, the "peptoid form" is used to refer to a mutant amino acid residue whose α-carbon substituent is on the nitrogen atom of that residue rather than on the α-carbon. Processes for preparing peptides in peptoid form are known in the art and are described, for example, in Simon RJ et al. , PNAS (1992) 89 (20), 9376-9371, and Howell DC, Trends Biotechnol. (1995) 13 (4), 132-134.

Homology modeling: Corresponding residues in other Cas9 orthologs are described in Zhang et al. , 2012 (Nature; 490 (7421): 556-60) and Chen et al. , 2015 (PLoS Comput Biol; 11 (5): e100248), can be identified by a computational protein-protein interaction (PPI) method that predicts interactions mediated by the domain-motif interface. PrePPI, a structure-based PPI prediction method, combines structural evidence with non-structural evidence using a Bayesian statistical framework. This method involves pairing query proteins and using structural alignments to identify structural representations that correspond to either of their experimentally determined structures or homology models. Structural alignment is also used to identify both near and far adjacent structures by considering global and local geometric relationships. Whenever two adjacent structures of a structural representation form a complex reported in a protein databank, this defines a template for modeling the interaction between these two query proteins. The model of the complex is created by superimposing representative structures on the corresponding adjacent structures in the mold. This method is described in Dey et al. , 2013 (Prot Sci; 22: 359-66).

For the purposes of the present invention, amplification means any method utilizing primers and polymerases capable of replicating the target sequence with sufficient fidelity. Amplification can be performed with natural or recombinant DNA polymerases such as TaqGold ™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. The preferred amplification method is PCR.

In some embodiments, the invention relates to vectors. As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. Replicons, such as plasmids, phages, or cosmids, can be inserted with another DNA segment to replicate this inserted segment. In general, a vector can replicate when attached to the appropriate control element. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another bound nucleic acid. Vectors are, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, without free ends (eg, cyclic). ) Nucleic acid molecules; Nucleic acid molecules containing DNA, RNA, or both; and various other polynucleotides known in the art. One type of vector is a "plasmid", which is, for example, a circular double-stranded DNA loop into which additional DNA segments can be inserted by standard molecular cloning techniques. Another type of vector is a viral vector, which is for packaging into viruses (eg, retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-related viruses (AAV)). Virus-derived DNA or RNA sequences are present in the vector. Viral vectors also include polynucleotides carried by the virus for transfection into host cells. Certain vectors are capable of self-renewal in the introduced host cell (eg, bacterial and episomal mammalian vectors with a bacterial origin of replication). Other vectors (eg, non-episome mammalian vectors) integrate into the host cell's genome when introduced into the host cell, thereby replicating with the host genome. In addition, certain vectors can induce the expression of functionally linked genes. Such vectors are referred to herein as "expression vectors." Common expression vectors useful for recombinant DNA technology are often in the form of plasmids.

The recombinant expression vector can contain the nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, i.e., the recombinant expression vector is functionally linked and expressed to the nucleic acid sequence to be expressed. Includes one or more regulatory elements that can be selected based on the host cell used in. In a recombinant expression vector, "functionally linked" means that the nucleotide sequence of interest allows expression of the nucleotide sequence (eg, in an in vitro transcription / translation system or in a host cell into which the vector has been introduced). It shall mean that it was connected to the adjustment element so as to. For recombinant and cloning methods, US Patent Application No. 10/815 published as US Patent Application Publication No. 2004-0171156 A1 on September 2, 2004, the entire disclosure of which is incorporated herein by reference. , No. 730.

Aspects of the invention relate to chimeric RNAs and bicistron vectors for Cas9. Bicistron expression vectors for chimeric RNA and Cas9 are preferred. In general, and especially in this embodiment, Cas9 is preferably driven by the CBh promoter. The chimeric RNA can preferably be driven by the Pol III promoter, eg, the U6 promoter. Ideally, the two promoters would be combined. The chimeric guide RNA typically consists of a 20 bp guide sequence (Ns), which can be linked to the tracr sequence, which extends from the first "U" in the lower strand to the end of the transcript. .. The tracr sequence can be cleaved at various positions as shown. The guide and tracr sequences are separated by a tracr mate sequence, which can be GUUUUAGAGCUA. This tracr sequence may be followed by the loop sequence GAAA shown. Both of these are preferred examples. Applicants have demonstrated Cas9-mediated insertion deletions at the human EMX1 and PVALB loci by the SURVEYOR assay. ChiRNA is indicated by its "+ n" designation, and crRNA refers to a hybrid RNA in which the guide and tracr sequences are expressed as separate transcripts. Throughout this application, chimeric RNAs may also be referred to as single-guided or synthetic-guided RNAs (sgRNAs).

In some embodiments, a loop is provided for the guide RNA. This may be a stem loop or a tetraloop. The loop is preferably GAAA, but is not limited to this sequence, or just 4 bp in length. In fact, the preferred loop-forming sequence used for the hairpin structure is 4 nucleotides in length, most preferably having the sequence GAAA. However, longer or shorter loop sequences can be used and these can be alternative sequences. This sequence preferably comprises a nucleotide triplet (eg AAA) and additional nucleotides (eg C or G). Examples of loop-forming sequences include CAAA and AAAG. In practicing any of the methods disclosed herein, suitable vectors can be introduced into cells or embryos by one or more methods known in the art and are limited to such methods. Micro-injection, electroporation, sonoporation, microscopic gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofe Includes action, lipofection, impalefection, phototransfection, proprietary drug-promoted nucleic acid uptake, and delivery by liposomes, immunolipolips, virosomes, or artificial virions. In some methods, the vector can be introduced into the embryo by microinjection. One or more vectors can be introduced into the embryo's nucleus or cytoplasm. In some methods, one or more vectors can be introduced into cells by nucleofection.

The term "regulatory element" shall include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals such as polyadenylation signals and poly-U sequences). Such adjusting elements include, for example, Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include regulatory elements that induce constitutive expression of nucleotide sequences in many types of host cells, and regulatory elements that induce expression of nucleotide sequences in specific host cells alone (eg, tissue-specific regulatory sequences). including. Tissue-specific promoters are expressed primarily in the desired tissue of interest, such as muscle, neurons, bones, skin, blood, specific organs (eg liver, pancreas), or specific cell types (eg lymphocytes). Can be induced. Regulatory elements can also induce expression in a time-dependent manner, eg, cell cycle-dependent, or developmental stage-dependent, which induction may be tissue-specific or cell-type-specific. , Or it does not have to be so specific. In some embodiments, the vector is one or more pol III promoters (eg, one, two, three, four, five, or more pol III promoters), one or more pol II. Promoters (eg, one, two, three, four, five, or more pol II promoters), one or more pol I promoters (eg, one, two, three, four, 5 or more pol I promoters), or combinations thereof. Examples of the pol III promoter include, but are not limited to, the U6 promoter and the H1 promoter. Examples of the pol II promoter include, but are not limited to, the retrovirus Raus sarcoma virus (RSV) LTR promoter (optionally including RSV enhancer), the cytomegalovirus (CMV) promoter (optionally including CMV enhancer) [ See, for example, Boshard et al, Cell, 41: 521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. Further, the term "adjustment element" refers to an enhancer element such as WPRE; CMV enhancer; R-U5'segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8 (1), p. 466). -472,1988); SV40 enhancer; and intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78 (3), p. 1527-31, 1981) is also included. Those skilled in the art will appreciate that the design of an expression vector can vary depending on factors such as the selection of the host detail to be transformed, the desired expression level, and the like. The vector is introduced into a host cell, whereby a transcript, protein, or peptide containing a fusion protein or fusion peptide encoded by the nucleic acids described herein (eg, clustered, regularly spaced short pari). Ndrome repeat (CRISPR) transcripts, proteins, enzymes, mutants thereof, fusion proteins thereof, etc.) can be produced. With respect to regulatory sequences, all disclosures thereof are described in US Patent Application No. 10 / 491,026, which is incorporated herein by reference. Promoters are described in PCT Publication International Publication No. 2011/028929 and US Patent Application No. 12 / 511,940, each of which is incorporated herein by reference in its entirety.

Vectors can be designed for expression of CRISPR transcripts (eg, nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, the CRISPR transcript can be expressed in bacterial cells such as Escherichia coli, insect cells (using a baculovirus expression vector), yeast cells, or mammalian cells. Suitable host cells include Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro using, for example, a T7 promoter regulatory sequence and T7 polymerase.

Vectors can be introduced into prokaryotes or prokaryotic cells for proliferation. In some embodiments, the prokaryote is an intermediate vector (eg, one of the viral vector packaging systems) to amplify a copy of the vector introduced into the eukaryote or in the production of the vector introduced into the eukaryote. It is used as a part to amplify the plasmid). In some embodiments, the prokaryote provides a source of one or more proteins for amplifying a copy of the vector to express one or more nucleic acids, eg, for delivery to a host cell or host organism. Used to do. Expression of proteins in prokaryotic organisms is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters that induce the expression of fusion or non-fusion proteins. The fusion vector adds a large number of amino acids to the amino terminus of the protein encoded therein, eg, a recombinant protein. Such fusion vectors have one or more purposes: (i) to increase the expression of the recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) as a ligand in affinity purification. By acting, it can help purify recombinant proteins. Often, in fusion expression vectors, proteolytic cleavage sites are introduced at the junction of the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes and their homologous recognition sequences include factors Xa, thrombin, and enterokinase. Examples of fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.), And pRA. ), Each of which fuses glutathione S-transferase (GST), maltose E-binding protein, or protein A to the target recombinant protein. Examples of suitable inducible non-fused E. coli expression vectors are pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studio et al., GENE EXPRESSION TECHNOLOGY: METHODS IN). ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987.EMBO J.6: 229-234), pMFa (Kuijan and Herskowt). -943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), And picZ (InVictorogen Corp, San Diego, Calif). In some embodiments, the vector uses a baculovirus expression vector to drive protein expression in insect cells. As baculovirus vectors that can be used for protein expression in cultured insect cells (eg, SF9 cells), the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series ( Lucklow and Summers, 1989. Virus 170: 31-39).

In some embodiments, the vector can be used to drive expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the regulatory function of the expression vector is typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Simian virus 40, and other viruses disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, eg, Chapters 16 and 17 of Sambrook, et al. , MOLECULAR Cloning: A LABORATORY MANUAL. 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. et al. Y. , 1989.

In some embodiments, the recombinant mammalian expression vector is capable of preferentially inducing nucleic acid expression in a particular cell type (eg, tissue-specific regulatory elements are used for nucleic acid expression). .. Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoters (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymph-specific promoters (Calame and Eaton, 1988.). Adv. Immunol. 43: 235-275), in particular the T cell receptor promoter (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and the immunoglobulin promoter (Baneiji, et al., 1983. Cell). 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), a neuron-specific promoter (eg, the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. 5477), a pancreas-specific promoter (Eld, et al., 1985. Science 230: 912-916), and a mammalian gland-specific promoter (eg, milk why promoter; US Pat. No. 4,873,316; And European Patent Application No. 264,166). The developmentally regulated promoters also include, for example, the murine hox promoter (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tiggman, 1989. Genes Dev. 3: 537-546). To do. These prokaryotic and eukaryotic vectors are described in US Pat. No. 6,750,059, the entire disclosure of which is incorporated herein by reference. Other embodiments of the invention may relate to the use of viral vectors, for which the entire disclosure of such viral vectors is incorporated herein by reference in US Patent Application No. 13/092,085. It is described in the specification. Tissue-specific regulatory elements are known in the art and are described in this regard in US Pat. No. 7,776,321, the entire disclosure of which is incorporated herein by reference. In some embodiments, the regulatory element is functionally linked to one or more elements of the CRISPR system to drive the expression of one or more elements of the CRISPR system. In general, CRISPR (Crustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDR (Sparer Interspired Direct Repeats), constitutes a family of DNA loci that are usually specific for a particular bacterial species. The CRISPR locus is a different class of short sequence repeats (SSRs) found in E. coli (SSR: short sequence repeat) (Ishino et al., J. Bacteriol., 169: 5249-5433 [1987]; and Includes Nakata et al., J. Bacteriol., 171: 3553-3556 [1989]) and related genes. Similar interspersed SSRs have also been identified in Haloferrax medialanei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (Groenen et al., Mol. -1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5: 254-263 [1999]; Massepohl et al., Biochim. Biophys. Acta 1307: 26-30 [1996]; and Mojica et al. , Mol. Microbiol., 17: 85-93 [1995]). The CRISPR locus is typically called short-regularly spaced repeats (SRSR), which, unlike other SSRs, has a repeat structure (Janssen et al., OMICS J. INTeg. Biol. , 6: 23-33 [2002]; and Mojica et al., Mol. Microbiol., 36: 244-246 [2000]). In general, this iteration is a short chain element that occurs in clusters that are regularly spaced by unique intervening sequences of substantially constant length (Mojica et al., [2000], supra). The repeats are highly conserved among the strains, but the number of interspersed repeats and the sequence of the spacer regions are typically different from strain to strain (van Embden et al., J. Bacteriol., 182: 2393-. 2401 [2000]). The CRISPR locus is, but is not limited to, Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halo-Aucula, Halocarcula. Methanobacterium, Metanococcus, Metanosarcina, Metanopyrus, Pyrococcus, Pyrococcus, Pyrococcus, Pyrococcus, Pyrococcus, Pyrococcus, Pyrococcus, Pyrococcus, Pyrococcus The genus Mycobacterium, the genus Streptomyces, the genus Aquifex, the genus Porphyromonas, the genus Chlorobium, the genus Chlorobium, the genus Thermus, the genus Termus, the genus Basilus ), Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Fusobacterium, Azoarcus, Azarcus, Azarcus Neisseria, Nitrosomonas, Desulfovivrio, Geobacter, Myxococcus, Campylobacter, Campylobacter, Warinella, Warinella, Warinella Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Photobacterium, Salmonella, Salmonella It has been identified in more than 40 prokaryotes, including as), Yersinia, Treponema, and Thermotogae (eg, Jansen et al. , Mol. Microbiol. , 43: 1565-1575 [2002]; and Mojica et al. , [2005]).

In general, a "nucleic acid targeting system", when used herein, collectively encodes a nucleic acid targeting Cas (effector) protein and a guide RNA, including a crRNA sequence and a trans-activated CRISPR / Cas system RNA (tracrRNA) sequence. Derives expression or activity of nucleic acid targeting CRISPR-related (“Cas”) genes (also referred to herein as effector proteins), including sequences to be produced, or other sequences and transcripts from the nucleic acid targeting CRISPR locus. Refers to transcripts and other elements involved in this. In some embodiments, one or more elements of the nucleic acid targeting system are derived from the V / VI nucleic acid targeting CRISPR system. In some embodiments, one or more elements of the nucleic acid targeting system are derived from a particular organism, including the endogenous nucleic acid targeting CRISPR system. In general, nucleic acid targeting systems are characterized by elements that facilitate the formation of nucleic acid targeting complexes at the site of the target sequence. In the context of the formation of a nucleic acid targeting complex, "target sequence" refers to a sequence in which the guide sequence is designed to be complementary to it, where the hybridization between the target sequence and the guide RNA is DNA or. Promotes the formation of RNA targeting complexes. Full complementarity is not always necessary if there is sufficient complementarity to generate hybridization and promote the formation of nucleic acid targeting complexes. The target sequence can include RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be within the organelles of eukaryotic cells, such as mitochondria or chloroplasts. A sequence or template that can be used for recombination to a target locus containing a target sequence is referred to as an "editing template" or "editing RNA" or "editing sequence". In aspects of the invention, the exogenous template RNA can be referred to as the editing template. In some aspects of the invention, the recombination is homologous recombination.

Typically, in the context of an endogenous nucleic acid targeting system, a nucleic acid targeting complex, including a guide RNA that hybridizes to a target sequence and forms a complex with one or more nucleic acid targeting effector proteins, is formed. And in or near the target sequence (eg, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more) Cleavage of one or both RNA strands occurs. In some embodiments, one or more vectors driving the expression of one or more elements of the nucleic acid targeting system are introduced into the host cell, and when the elements of the nucleic acid targeting system are expressed, the nucleic acid at one or more target sites. The formation of the targeting complex is guided. For example, nucleic acid targeting effector proteins and guide RNAs may be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined into a single vector, with one or more additional vectors not included in the first vector of nucleic acid targeting systems. Provide any component. Nucleic acid targeting elements combined into a single vector have one element located 5'side ("upstream" of it) or 3'side ("downstream") of the second element. It may be arranged in any suitable orientation, such as being positioned. The coding sequence of one element may be located on the same chain or reverse chain as the coding sequence of the second element, and may be oriented in the same or opposite direction. In some embodiments, a single promoter is integrated within one or more intron sequences (eg, each in a different intron, two or more in at least one intron, or all). It drives the expression of transcripts encoding nucleic acid targeting effector proteins and guide RNAs (in a single intron). In some embodiments, the nucleic acid targeting effector protein and the guide RNA are operably linked to the same promoter and may be expressed from there.

In general, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and derive sequence-specific binding of the nucleic acid targeting complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is approximately 50%, 60%, 75%, 80% when optimally aligned using a suitable alignment algorithm. , 85%, 90%, 95%, 97.5%, 99% or more, or higher. Optimal alignment can be determined using any algorithm suitable for sequence alignment, examples of which are non-limiting examples: Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burroughs-Wheeler transformation based algorithm. Available in (eg, Burroughs Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), SOAP (soap.genomics.org.org.c). (Available in .net). In some embodiments, the guide sequences are approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2. 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer, or longer. In some embodiments, the guide sequence is about 75. , 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length or less, or shorter. The ability of the guide sequence to induce sequence-specific binding of the nucleic acid targeting complex to the target sequence. Can be evaluated by any suitable algorithm, for example, to host cells having the corresponding target sequence, including the guide sequence to be tested, with sufficient components of the nucleic acid targeting system to form the nucleic acid targeting complex. And may be provided, for example, by transfection of a vector encoding a component of the nucleic acid targeting CRISPR sequence, followed by preference within or near the target sequence, such as by a Surveyor algorithm as described herein. Cleavage of the target polynucleotide sequence (or a sequence in the vicinity thereof) can be evaluated for the target sequence, the components of the nucleic acid targeting complex including the guide sequence to be tested, and the test. A control guide sequence different from the guide sequence can be provided and determined in vitro by comparing the binding or cleavage rate in or near the target sequence between the reactions of those test guide sequences and the control guide sequence. Yes, other assays are possible and will be recalled by those skilled in the art.

The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a gene transcript or mRNA.

In some embodiments, the target sequence is a sequence within the genome of a cell.

In some embodiments, the guide sequence is selected so that the degree of secondary structure within the guide sequence is reduced. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of such an algorithm is mFold as described by Zuker and Steigler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is an online web server RNAfold that uses a centroid structure prediction algorithm developed at the Institute for Theoretical Chemistry at the University of Vienna (eg, AR Grubber et al.). , 2008, Cell 106 (1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62). For further algorithms, US Provisional Patent Application No. 61 / 863,080 (incorporated herein by reference) can be referred to.

In some embodiments, recombinant templates are also provided. The recombinant template can be a component of another vector as described herein, either contained in a separate vector or provided as a separate polynucleotide. In some embodiments, the recombinant template is designed to act as a template in homologous recombination, such as in or near a target sequence that is nicked or cleaved by a nucleic acid targeting effector protein as part of a nucleic acid targeting complex. Will be done. The template polynucleotide may be of any suitable length, such as about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 nucleotides or longer, or longer. In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide containing the target sequence. When optimally aligned, the template polynucleotide is one or more nucleotides of the target sequence (eg, about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90). , 100 nucleotides or more or longer). In some embodiments, when the template sequence and the polynucleotide containing the target sequence are optimally aligned, the closest nucleotide of the template polynucleotide is approximately 1, 5, 10, 15, 20, 25 from the target sequence. , 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000 nucleotides or less, or more.

In some embodiments, the nucleic acid targeting effector protein is one or more heterologous protein domains (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 in addition to the nucleic acid targeting effector protein). It is part of a fusion protein that contains more than one or more domains). In some embodiments, the CRISPR effector protein / enzyme has one or more heterologous protein domains (eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more in addition to the CRISPR enzyme). , Or more domains) are part of the fusion protein. The CRISPR enzyme fusion protein may contain any additional protein sequence and a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, receptor gene sequences, and the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repressive activity, transcriptional release. Examples include protein domains having one or more of factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), blue-green peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, Examples include autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyanide fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes include DNA molecules or others, including maltose-binding protein (MBP), S-tag, Lex A DNA-binding domain (DBD) fusion, GAL4 DNA-binding domain fusion, and simple herpesvirus (HSV) BP16 protein fusion. It can be fused to a gene sequence encoding a protein or fragment of a protein that binds to a cell molecule of. Additional domains that may form part of the fusion protein, including the CRISPR enzyme, are described in US Patent Application Publication No. 20110059502, which is incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the localization of the target sequence.

In some embodiments, the CRISPR enzyme can form a component of the inducing system. The inducibility of this system will allow spatiotemporal control of gene editing or gene expression using certain energy forms. This form of energy may include, but is not limited to, electromagnetic radiation, sound wave energy, chemical energy, and thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule 2-hybrid transcriptional activation systems (FKBP, ABA, etc.), or photoinduction systems (phytochrome, LOV domain, or cryptochrome). Can be mentioned. In one embodiment, the CRISPR enzyme can be part of a photo-induced transcriptional effector (LITE) that induces sequence-specific changes in transcriptional activity. Light components may include CRISPR enzymes, photoresponsive cytochrome heterodimers (eg, from Arabidopsis thaliana), and transcriptional activation / repressor domains. Further examples of the inducible DNA-binding protein and its use are described in US Provisional Patent Application Nos. 61/736465 and 61 / 721,283, respectively, of which the entire disclosure is incorporated herein by reference. International Publication No. 2014/018423 and US Patent No. 8889418, US Patent No. 8895308, US Patent Application Publication No. 20140186919, US Patent Application Publication No. 20140242700, US Patent Application Publication No. It is described in the specification of 20140273234, the specification of US Patent Application Publication No. 20140335620, and the pamphlet of International Publication No. 2014093635.

In some embodiments, the invention is transcribed from one or more polynucleotides, eg, or one or more vectors as described herein, one or more transcripts thereof, and / or from it. Provided are methods that include the step of delivering a substance or protein to a host cell. In some embodiments, the invention further provides cells produced by such methods and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a nucleic acid targeting effector protein combined with (and optionally complexed with) a guide RNA is delivered to the cell. Conventional virus-based and non-virus-based gene transfer methods can be used for the introduction of nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of the nucleic acid targeting system to cells in culture or to cells in the host organism. Non-viral vector delivery systems include DNA plasmids, RNA (eg, transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles, such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either an episomal genome or an integrated genome after delivery to cells. For a review of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel & Fergner, TIBTECH 11: 211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); TIBTECH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Ristorante Neuroscience 1995); Kremer & Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Haddada et al. , In Current Topics in Microbiology and Immunology, Doerfler and Boehm (eds) (1995); and Yu et al. , Gene Therapy 1: 13-26 (1994).

Non-viral methods of delivering nucleic acids include lipofection, nucleofection, microinjection, microscopic guns, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial virions, and drug-promoted DNA uptake. Is included. Lipofection is described, for example, in US Pat. Nos. 5,049,386, 4,946,787; and 4,897,355), and lipofection reagents are commercially available. (For example, Transfectam ™ and Lipofection ™). Cationic and triglyceride suitable for efficient receptor recognition lipofection of polynucleotides include those of Fergner, WO 91/17424; Pamphlet 91/16024. Delivery may be to cells (eg, in vitro or exovivo administration) or to target tissue (eg, in vivo).

Preparation of lipid: nucleic acid complexes is well known to those of skill in the art, including targeted liposomes such as immunolipid complexes (eg, Crystal, Science 270: 404-410 (1995); Blaese et al., Cancer Gene Ther. .2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene. Therapy 2: 710-722 (1995); Ahmad et al., Cancer Res. 52: 4817-4820 (1992); US Pat. Nos. 4,186,183, 4,217,344, No. 4,235,871 No. 4,261,975 No. 4,485,054 No. 4, 501,728 No. 4, 774, No. 085, 4,837,028, and 4,946,787).

Delivery of nucleic acids using RNA or DNA virus-based systems utilizes a highly evolved process of targeting the virus to specific cells in the body and transporting the viral payload to the nucleus. The viral vector may be administered directly to the patient (in vivo) or may be used to treat the cells in vitro, and the modified cells may be optionally administered to the patient (exo vivo). Traditional virus-based systems can include retroviruses, lentiviruses, adenoviruses, adeno-associated and herpes simplex virus vectors for gene transfer. Retrovirus, lentivirus, and adeno-associated virus gene transfer methods allow integration in the host genome, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

Retrovirus tropism can be altered by uptake of exogenous enveloped proteins, which can expand the potential target population of target cells. A lentiviral vector is a retroviral vector capable of transducing or infecting non-dividing cells and typically producing a high viral titer. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors consist of cis-acting long-term repeats with the ability to package foreign sequences up to 6-10 kb. A minimal cis-acting LTR is sufficient for vector replication and packaging, which can then be used to integrate the therapeutic gene into target cells, resulting in permanent transgene expression. Widely used retrovirus vectors are based on murine leukemia virus (MuLV), tenaga monkey leukemia virus (GaLV), monkey immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. Included (eg, Buchcher et al., J. Virus. 66: 2731-2739 (1992); Johann et al., J. Virus. 66: 1635-1640 (1992); Somenerfeld et al., Virus. 176). : 58-59 (1990); Wilson et al., J. Virus. 63: 2374-2378 (1989); Miller et al., J. Virus. 65: 2220-2224 (1991); PCT / US94 / 05700. See specification). Adenovirus-based systems can be used for applications where transient expression is preferred. Adenovirus-based vectors can exhibit extremely high transduction efficiencies in many cell types and do not require cell division. High titers and expression levels have been obtained with such vectors. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors can also be used, for example, in in vitro production of nucleic acids and peptides, and for in vivo and exobibo gene therapy procedures, for transduction of cells with target nucleic acids (eg, West et). al., Virology 160: 38-47 (1987); US Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5: 793-801 (1994); Muzyczka, See J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors has been made in numerous publications, eg, US Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5: 3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4: 2072-2081 (1984); Hermonat & Muzyczka, PNAS 81: 6466-6470 (1984); And Samulski et al., J. Virol. 63: 03822-3828 (1989).

Substantiation of Delivery The present invention relates to at least one component of a CRISPR complex delivered by at least one nanoparticle complex, such as RNA. In some embodiments, the invention is transcribed from one or more polynucleotides, eg, or one or more vectors as described herein, one or more transcripts thereof, and / or from it. Provided are methods that include the step of delivering a substance or protein to a host cell. In some embodiments, the invention further provides cells produced by such methods and animals comprising or produced from such cells. In some embodiments, the CRISPR enzyme combined with the guide sequence (and optionally complexed with it) is delivered to the cell. Conventional virus-based and non-virus-based gene transfer methods can be used for the introduction of nucleic acids in mammalian cells or target tissues. Using such a method, the nucleic acid encoding a component of the CRISPR system can be administered to cells in culture or to cells in the host organism. Non-viral vector delivery systems include DNA plasmids, RNA (eg, transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles, such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either an episomal genome or an integrated genome after delivery to cells. For a review of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel & Fergner, TIBTECH 11: 211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); TIBTECH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Ristorante Neuroscience 1995); Kremer & Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Haddada et al. , In Current Topics in Microbiology and Immunology Doerfler and Boehm (eds) (1995); and Yu et al. , Gene Therapy 1: 13-26 (1994).

Non-viral delivery methods of nucleic acids include lipofection, microinjection, microscopic guns, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial virions, and drug-promoted DNA uptake. Lipofection is described, for example, in US Pat. Nos. 5,049,386, 4,946,787; and 4,897,355), and lipofection reagents are commercially available. (For example, Transfectam ™ and Lipofection ™). Cationic and triglyceride suitable for efficient receptor recognition lipofection of polynucleotides include those of Fergner, WO 91/17424; Pamphlet 91/16024. Delivery may be to cells (eg, in vitro or exovivo administration) or to target tissue (eg, in vivo).

Preparation of lipid: nucleic acid complexes is well known to those of skill in the art, including targeted liposomes such as immunolipid complexes (eg, Crystal, Science 270: 404-410 (1995); Blaese et al., Cancer Gene Ther. .2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene. Therapy 2: 710-722 (1995); Ahmad et al., Cancer Res. 52: 4817-4820 (1992); US Pat. Nos. 4,186,183, 4,217,344, No. 4,235,871 No. 4,261,975 No. 4,485,054 No. 4, 501,728 No. 4, 774, No. 085, 4,837,028, and 4,946,787).

Delivery of nucleic acids using RNA or DNA virus-based systems utilizes a highly evolved process of targeting the virus to specific cells in the body and transporting the viral payload to the nucleus. The viral vector may be administered directly to the patient (in vivo) or may be used for the treatment of cells in vitro, and the modified cells may be optionally administered to the patient (ex vivo). Traditional virus-based systems can include retroviruses, lentiviruses, adenoviruses, adeno-associated and herpes simplex virus vectors for gene transfer. Retrovirus, lentivirus, and adeno-associated virus gene transfer methods allow integration in the host genome, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

Retrovirus tropism can be altered by uptake of exogenous enveloped proteins, which can expand the potential target population of target cells. A lentiviral vector is a retroviral vector capable of transducing or infecting non-dividing cells and typically producing a high viral titer. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors consist of cis-acting long-term repeats with the ability to package foreign sequences up to 6-10 kb. A minimal cis-acting LTR is sufficient for vector replication and packaging, which can then be used to integrate the therapeutic gene into target cells, resulting in permanent transgene expression. Widely used retrovirus vectors are based on murine leukemia virus (MuLV), tenaga monkey leukemia virus (GaLV), monkey immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. Included (eg, Buchcher et al., J. Virus. 66: 2731-2739 (1992); Johann et al., J. Virus. 66: 1635-1640 (1992); Somenerfeld et al., Virus. 176). : 58-59 (1990); Wilson et al., J. Virus. 63: 2374-2378 (1989); Miller et al., J. Virus. 65: 2220-2224 (1991); PCT / US94 / 05700. See specification).

In another embodiment, Cocal becyclovirus envelope pseudoretroviral vector particles are contemplated (eg, US Patent Application Publication No. 20120164118 transferred to Fred Hutchinson Cancer Research Center). See the specification). The cocalvirus belongs to the genus Vesculovirus and is the causative agent of bullous stomatitis in mammals. The cocal virus was initially isolated from mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 25: 236-242 (1964)) and is an insect, bovine in Trinidad, Brazil, and Argentina. , And the infection has been identified from horses. Many of the becycloviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-mediated. Antibodies to the becyclovirus are common in people living in rural areas, where the virus is endemic and laboratory infectious; infections in humans usually result in influenza-like symptoms. The cocalvirus envelope glycoprotein shares 71.5% identity with VSV-G Indiana at the amino acid level, and a phylogenetic comparison of the envelope genes of becyclovirus shows that cocalvirus is among the becycloviruses. It is shown to be the most closely related, although serologically different from the VSV-G Indiana strain. Jonkers et al. , Am. J. Vet. Res. 25: 236-242 (1964) and Travasos da Rosa et al. , Am. J. Tropical Med. & Hygiene 33: 999-1006 (1984). The cocalbecyclovirus envelope pseudoretroviral vector particles can include, for example, the retrovirus Gag, Pol, and / or one or more accessory proteins and the cocalbecyclovirus envelope protein, lentivirus, alpha retrovirus, beta. Retroviruses, gamma retroviruses, delta retroviruses, and epsilon retrovirus vector particles can be included. Within certain aspects of these embodiments, the Gag, Pol, and accessory proteins are those of lentivirus and / or gammaretrovirus. The present invention comprises or is essentially an exogenous nucleic acid molecule encoding a CRISPR system, eg, a promoter and a CRISPR-related (Cas) protein (estimated nuclease or helicase protein), eg, a nucleic acid molecule encoding Cas9, and a terminator. A first cassette and promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator, and two or more, preferably vector packaging size limits. Up to, for example, a total of 5 cassettes (including the first cassette) (eg, each cassette is generally a promoter-gRNA1-terminator, a promoter-gRNA2-terminator ... promoter-gRNA (N)). -AAV containing or consisting of multiple cassettes containing or consisting of a promoter (in the formula, N is represented by the number of insertable numbers that is the upper limit of the packaging size limit of the vector), or an AAV that is essentially derived from it. Two or more individual rAAVs, each containing one or more cassettes of the CRISPR system, eg, a first rAAV is a nucleic acid molecule encoding a promoter and Cas, eg Cas (Cas9). And a first cassette containing or essentially becoming a terminator, and a second rAAV containing or essentially a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator. Multiple four cassettes (eg, each cassette is generally a promoter-gRNA1-terminator, a promoter-gRNA2-terminator ... promoter-gRNA (N) -terminator (in the formula, N is the packaging size of the vector). Provided is an rAAV that includes (represented as an insertable number) that is the upper limit of the limit. Since rAAV is a DNA virus, the nucleic acid molecule in the discussion herein with respect to AAV or rAAV is advantageously DNA. The promoter, in some embodiments, is advantageously the human synapsin I promoter (hSyn). Further methods of delivering nucleic acids to cells are known to those of skill in the art. See, for example, US Patent Application Publication No. 20030087817, which is incorporated herein by reference.

In some embodiments, the host cell is transiently or non-transiently transfected with one or more of the vectors described herein. In some embodiments, the cell is transfected as it is naturally present in the subject. In some embodiments, the cells to be transfected are harvested from the subject. In some embodiments, the cells are derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MALT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC- 3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI- 231, HB56, TIB55, Jarkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryo fibroblasts, 3T3 Switzerland, 3T3-L1, 132-d5 human fetal fibroblasts; 10. 1 mouse fibroblast, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cell, BEAS-2B, bEnd .. 3, BHK-21, BR293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2 , EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jarkat, JY cells, K562 Cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II , MDCK II, MOR / 0.2R, MONO-MAC6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM- 1, NW-145, OPCN / OPCT cell line, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 Included are cell lines, U373, U87, U937, VCaP, Velo cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines are available from a variety of sources known to those of skill in the art (see, eg, the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, cells transfected with one or more of the vectors described herein are used to establish novel cell lines containing one or more vector-derived sequences. In some embodiments, the components of the CRISPR system as described herein are transiently transfected (eg, by transient transfection of one or more vectors, or transfection with RNA). Using cells that have been affected and modified by the activity of the CRISPR complex, new cell lines are established that include cells that contain the modification but lack any other exogenous sequence. In some embodiments, using cells that have been transiently or non-transiently transfected with one or more of the vectors described herein, or cell lines derived from such cells, 1 One or more test compounds are evaluated.

In some embodiments, non-human transgenic animals or transgenic plants are made using one or more of the vectors described herein. In some embodiments, the transgenic animal is a mammal such as a mouse, rat, or rabbit. Methods for producing transgenic animals and plants are known in the art and generally begin with cell transfection methods, such as those described herein. In another embodiment, a fluid delivery device comprising an array of needles (see, eg, US Patent Application Publication No. 20112030839 transferred to the Fred Hutchinson Cancer Research Center) is a solid tissue. Can be intended for delivery of CRISPR Cas to. The device of U.S. Patent Application Publication No. 20112030839 for delivering fluid to solid tissue is composed of a plurality of needles arranged in an array; a plurality of reservoirs each communicating with one of the plurality of needles. And; may include a plurality of actuators operably coupled to each of the plurality of reservoirs and configured to control fluid pressure in the reservoirs. In certain embodiments, each of the plurality of actuators can include one of a plurality of plungers, the first end of each of the plurality of plungers is received in each one of the plurality of reservoirs, and specific. In another embodiment, the plungers of the plurality of plungers are operably coupled together at their second ends and can be pushed down at the same time. Certain yet another embodiment may include a plunger drive configured to push down all of the plungers at selectively changeable speeds. In another embodiment, each of the plurality of actuators can include one of a plurality of fluid delivery paths having a first end and a second end, each of the plurality of fluid delivery paths. The end of one is coupled to each one of the plurality of reservoirs. In other embodiments, the device can include a fluid pressure source, and each of the plurality of actuators comprises a fluid coupling between the fluid pressure source and each one of the plurality of reservoirs. In a further embodiment, the fluid pressure source may include at least one of a compressor, a vacuum accumulator, a perturbing pump, a master cylinder, a microfluidic pump, and a valve. In another embodiment, each of the needles may include multiple ports arranged along its length.

Delivery to the Kidney Delivery methods to the kidney are summarized as follows.

Delivery to the Brain Options for delivery to the brain include encapsulating CRISPR enzymes and guide RNAs in either form of DNA or RNA in liposomes and conjugating them to molecular trojans to cross the blood-brain barrier (BBB). Includes delivery. Molecular Trojans have been shown to be effective in delivering B-gal expression vectors to the brains of non-human primates. The same technique can be used to deliver a vector containing a CRISPR enzyme and a guide RNA. For example, Xia CF and Boado RJ, Pardrigge WM (“Antibody-mediated targeting of siRNA via the biotin receptor)” Mol Pharm. 2009 May-Jun; 6 (3): 747-51. Doi: 10.102 / mp800194) was used in combination with receptor-specific monoclonal antibody (mAb) and avidin-biotin technology in culture and in vivo. It describes how small interfering RNAs (siRNAs) can be delivered to cells in. The authors also observed RNAi effects in distant sites such as the brain in vivo after intravenous administration of the targeted siRNA because the binding between the targeting mAb and siRNA is stable with avidin-biotin technology. Also report that it will be done.

Zhang et al. (Mol Ther. 2003 Jan; 7 (1): 11-8)) consisted of 85 nm pegged immune plasmids targeted to red-throated monkey brain in vivo with a monoclonal antibody (MAb) against the human insulin receptor (HIR). It describes how an expression plasmid encoding a reporter such as luciferase was encapsulated inside the "artificial virus". HIRMAb allows liposomes carrying exogenous genes to undergo transcytosis across the blood-brain barrier and endocytosis across the nerve cell membrane after intravenous injection. The level of luciferase gene expression in the brain was 50-fold higher in rhesus monkeys compared to rats. Extensive neuronal expression of the β-galactosidase gene in the primate brain has been demonstrated by both histochemistry and confocal microscopy. The authors show that this technique makes reversible adult transgenics feasible in 24 hours. Therefore, the use of immunoliposomes is preferred. When used in combination with antibodies, specific tissue or cell surface proteins are targeted.

Delivery of HSCs to hematopoietic stem cells and their editing; and detailed conditions The term "hematopoietic stem cells" or "HSC" gives rise to cells considered HSCs, such as all other blood cells, and is derived from the mesoderm; It is intended to broadly include blood cell cells located in the red pulp contained in the central part of the bone. The HSCs of the invention include small size, lack of line markers, and markers belonging to a series of differentiation clusters, such as: CD34, CD38, CD90, CD133, CD105, CD45, and also c-kit (stem cell factor). Includes cells with the hematopoietic stem cell phenotype identified by the receptor). Hematopoietic stem cells are negative for markers used to detect differentiation lineage determination and are therefore called Lin-; and during their purification by FACS, a number of up to 14 different mature blood lineage markers, eg, bone marrow cells for humans. CD13 and CD33, CD71 of red blood cells, CD19 of B cells, CD61 of macronuclear cells, etc .; and B220 of B cells (mouse CD45), Mac-1 of monocytes (CD11b / CD18), Gr-1 of granulocytes. , Erythrocyte lineage Ter119, T cell Il7Ra, CD3, CD4, CD5, CD8, etc. Mouse HSC markers: CD34lo / −, SCA-1 +, Thy1.1 + / lo, CD38 +, C-kit +, lin-, and human HSC markers: CD34 +, CD59 +, Thy1 / CD90 +, CD38lo / −, C-kit / CD117 +, And lin-. HSCs are identified by markers. Thus, in the embodiments discussed herein, the HSC can be a CD34 + cell. The HSC may also be a hematopoietic stem cell that is CD34- / CD38-. Stem cells that may lack c-kit on the cell surface that are considered HSCs in the art are within the scope of the invention, and CD133 + cells are also considered HSCs in the art.

The CRISPR-Cas (eg Cas9) system can be engineered to target one or more loci of the HSC. Advantageously, eukaryotic cells and especially mammalian cells, such as human cells, such as HSC codon-optimized Cas (eg Cas9) proteins, and sgRNAs that target one or more loci of HSC, such as gene EMX1. Can be prepared. These may be delivered by particles. Particles can be formed by mixing a Cas (eg Cas9) protein with an sgRNA. The sgRNA and Cas (eg Cas9) protein mixture may contain or be mixed with a mixture consisting of, for example, surfactants, phospholipids, biodegradable polymers, lipoproteins and alcohols. Well, this can form particles containing sgRNA and Cas (eg Cas9) protein. The present invention includes making particles as such and particles from such methods and their use.

More generally, particles are formed using an efficient process. First, the Cas (eg Cas9) protein and the sgRNA targeting the gene EMX1 or control gene LacZ are preferably preferably in sterile nuclease-free buffer, eg 1x PBS, eg 3: 1-1: 1. At a suitable temperature, such as 15-30 ° C, for example 20-25 ° C, for example room temperature, for a suitable time, such as 15-45, for example 30 minutes, in a molar ratio of 3 or 2: 1 to 1: 2 or 1: 1. Can be mixed together over. Apart from that, surfactants such as cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); phospholipids such as dimyristylphosphatidylcholine (DMPC); biodegradable polymers such as ethylene. Glycol polymers or PEGs and lipoproteins such as low density lipoproteins such as cholesterol, or particle components containing them, alcohols, preferably C1-6 alkyl alcohols such as methanol, ethanol, isopropanol, for example 100. May be soluble in% ethanol. These two solutions can be mixed together to form particles containing a Cas (eg, Cas9) -sgRNA complex. In certain embodiments, the particles may contain an HDR template. This may be particles co-administered with sgRNA + Cas (eg Cas9) protein-containing particles, or ie, in addition to contacting the HSC with the sgRNA + Cas (eg Cas9) protein-containing particles, the HSC contains an HDR template. Or contact the HSC with particles containing all of the sgRNA, Cas (eg Cas9) and HDR templates. The HDR template may be administered by a separate vector, whereby in the first example the particles invade the HSC cells, and the separate vector also invades the cells, where the HSC genome is sgRNA + Cas (eg Cas9). And the HDR template is also present, thereby modifying the genomic locus by HDR; for example, this can result in mutation modification.

After particle formation, HSCs in 96-well plates can be transfected with 15 ug Cas (eg Cas9) protein per well. HSCs can be harvested 3 days after transfection to quantify the number of insertions and deletions (indels) at the EMX1 locus.

This illustrates how CRISPR-Cas (eg, Cas9) that targets one or more genomic loci of interest in HSC can be used to modify HSC. The modified HSC is in vivo, ie, organisms such as humans or non-human eukaryotes such as fish such as zebrafish, mammals such as primates, such as apes, chimpanzees, mackerel, rodents, etc. For example, mice, rabbits, rats, canine animals or dogs, domestic animals (cow / bovine, sheep / sheep, goats or pigs), birds or poultry, such as chickens, etc. It may be the HSC of the animal. The modified HSC may be an HSC in vitro, i.e., external to such organism. And the modified HSCs can be used in exovivos, i.e., one or more HSCs of such organisms can be obtained or isolated from the organism, even if one or more HSCs are optionally expanded. Often, one or more HSCs are contacted with, for example, one or more HSCs by a composition comprising CRISPR-Cas (eg Cas9) that targets one or more loci of HSCs. Modified by, for example, where the composition comprises or becomes essentially a mixture of sgRNA and Cas (eg Cas9) proteins, including surfactants, phospholipids, biodegradable polymers, lipoproteins and alcohols. Containing particles containing a CRISPR enzyme and one or more sgRNAs that target one or more loci of the HSC, such as particles obtained or can be obtained by mixing with a mixture of them (here). One or more sgRNAs target one or more loci of HSCs), optionally expand the modified HSCs obtained, and administer the resulting modified HSCs to the organism. In some cases, the isolated or obtained HSC may be derived from a first organism, such as an organism derived from the same species as the second organism, and the second organism administers the resulting modified HSC. It may be an organism, for example, the first organism may be a donor to the second organism (such as a blood relative as in the case of a parent or sibling). The modified HSC may have a genetic modification that addresses or alleviates or reduces the symptoms of the disease or condition of the individual or subject or patient. The modified HSC has a genetic modification having the HSC having one or more proteins, eg, a surface marker or protein more similar to that of the second organism, for example in the case of a first organism donor to a second organism. obtain. The modified HSC may have a genetic modification that stimulates the disease or condition of the individual or subject or patient and may be re-administered to a non-human organism to prepare an animal model. The expansion of HSCs is within the scope of those skilled in the art from this disclosure and knowledge in the art, eg, Lee, "improved ex vivo expansion of adult by eliminating CUL4-mediated HOXB4 degradation. Hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4) "Blood. 2013 May 16; 121 (20): 4082-9. doi: 10.1182 / blood-2012-09-455204. See EPUB 2013 Mar 21.

Therefore, as shown to improve activity, sgRNAs may be pre-complexed with Cas (eg, Cas9) proteins before forming the entire complex as particles. Different components of different molar ratios known to facilitate delivery of nucleic acids to cells (eg 1,2-dioleoil-3-trimethylammonium propane (DOTAP), 1,2-ditetradecanoyl-sn- Formulations may be made with glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG), and cholesterol), eg, DOTAP: DMPC: PEG: cholesterol molar ratios are DOTAP100, DMPC0, PEG0, cholesterol 0; or DOTAP90. , DMPC0, PEG10, cholesterol 0; or DOTAP90, DMPC0, PEG5, cholesterol 5, DOTAP100, DMPC0, PEG0, cholesterol 0. Accordingly, the present invention includes the step of mixing the sgRNA, the Cas (eg, Cas9) protein with the constituents forming the particles; and the particles from such a mixing step.

In a preferred embodiment, the particles containing the Cas (eg Cas9) -sgRNA complex mix the Cas (eg Cas9) protein with one or more sgRNAs together in an enzyme: guide RNA, preferably in a 1: 1 molar ratio. Can be formed by Separately, various components known to facilitate the delivery of nucleic acids (eg, DOTAP, DMPC, PEG, and cholesterol) are preferably dissolved in ethanol. These two solutions are mixed together to form particles containing the Cas (eg Cas9) -sgRNA complex. After the particles are formed, the Cas (eg Cas9) -sgRNA complex can be transfected into cells (eg HSC). Bar coding may be applied. Particles, Cas-9 and / or sgRNA can be barcoded.

The present invention, in certain embodiments, comprises a mixture of sgRNA and Cas (eg, Cas9) proteins containing or consisting essentially of surfactants, phospholipids, biodegradable polymers, lipoproteins and alcohols. Includes methods for preparing sgRNA- and -Cas (eg Cas9) protein-containing particles, including the step of mixing with. Certain embodiments include sgRNA- and -Cas (eg Cas9) protein-containing particles from this method. The present invention, in certain embodiments, is a method of modifying a target genomic locus, or an organism or non-human organism, by manipulating a target sequence at the target genomic locus, comprising the target genomic locus. A method [where sgRNA targets a genomic locus of interest] comprising contacting the cells with the particles; or by manipulating the target sequence at the genomic locus of interest to the genomic locus of interest, or organism. Alternatively, the use of the particles in a method of modifying a non-human organism, comprising contacting a cell containing the genomic locus of interest with the particle [where sgRNA targets the genomic locus of interest]. Including. In these embodiments, the genomic locus of interest is advantageously the genomic locus of HSC.

Therapeutic application considerations: A consideration in genome editing therapy is the selection of sequence-specific nucleases, such as variants of the Cas9 nuclease. Each nuclease variant can have a set of strengths and weaknesses inherent in it, many of which must be balanced so that the therapeutic benefit is maximized in the context of the treatment. So far, two therapeutic editing techniques with nucleases, namely gene disruption and gene modification, have shown remarkable promise. Gene disruption involves the creation of target indels in the genetic element by stimulating NHEJ and often results in loss-of-function mutations that are beneficial to the patient. In contrast, genetic modification directly restores disease-causing mutations using HDR and restores function while maintaining the physiological regulation of the modified element. HDR can also be used to insert a therapeutic transgene into a defined "safe harbor" locus in the genome to restore defective gene function. For a particular editorial therapy to be effective, a modification level high enough to reverse the disease symptoms in the target cell population must be achieved. This therapeutic modification "threshold" is determined by the fitness of the edited cells after treatment and the amount of gene product required to reverse the symptoms. In terms of fitness, treated cells may have three consequences from editing compared to their unedited counterparts: increased fitness, intermediate fitness, or decreased fitness. When fitness increases (eg, in the treatment of SCID-X1), modified hematopoietic progenitor cells selectively expand compared to their unedited counterpart. SCID-X1 is a disease caused by mutations in the IL2RG gene, whose function is required for the normal development of hematopoietic and lymphocyte lines [Leonard, W. et al. J. , Et al. Immunological reviews 138, 61-86 (1994); Kaushansky, K. et al. & Williams, W.W. J. Williams hematology, (McGraw-Hill Medical, New York, 2010)]. In clinical trials in patients who received SCID-X1 viral gene therapy, and in rare cases of spontaneous modification of SCID-X1 mutations, modified hematopoietic progenitor cells eliminated this developmental blockade and compared to their affected counterparts. By expanding, it may be possible to mediate therapies [Bousso, P. et al. , Et al. Proceedings of the National Academia of Sciences of the United States of America 97, 274-278 (2000); Hacein-Bay-Abina, S.A. , Et al. The New England journal of medicine 346, 1185-1193 (2002); Gaspar, H. et al. B. , Et al. Lancet 364,2181-2187 (2004)]. In this case, the edited cells have a selective advantage and can be amplified through expansion, even with a small number of edited cells, providing therapeutic benefit to the patient. In contrast, editing of other hematopoietic disorders such as chronic granulomatous disease (CGD) does not change the fitness of the edited hematopoietic progenitor cells and may increase the therapeutic modification threshold. CGD is usually caused by mutations in the gene encoding the phagocytic oxidase protein used by neutrophils to produce reactive oxygen species that kill pathogens [Mukherjee, S. et al. & Thrasher, A.M. J. Gene 525,174-181 (2013)]. The dysfunction of these genes does not affect the fitness or development of hematopoietic progenitor cells, only the ability of mature hematopoietic cell types to combat infection, so preferential expansion of edited cells in this disease It seems that there is no such thing. In fact, no selective predominance for gene-modified CGD cells has been observed in gene therapy trials, making long-term cell engraftment difficult [Malech, H. et al. L. , Et al. Proceedings of the National Academy of Sciences of the United States of America 94, 12133-12138 (1997); J. , Et al. Molecular therapy: the journal of the American Society of Gene Therapy 19, 2092-2101 (2011)]. Therefore, a significantly higher level of editing may be required for the treatment of diseases such as CGD, where editing results in an intermediate fitness advantage compared to diseases in which editing results in increased fitness for target cells. When editing imposes a fitness disadvantage, as in the case of the recovery function for tumor suppressor genes in cancer cells, the modified cells are defeated by their morbidity and the therapeutic benefit is lower than the editing rate. obtain. This latter class of disease can be particularly difficult to treat with genome editing therapy.

In addition to cell fitness, the amount of gene product needed to treat the disease also influences the lowest level of therapeutic genome editing to be achieved to reverse symptoms. Hemophilia B is a disease in which small changes in gene product levels can lead to large changes in clinical outcome. The disease is caused by mutations in a gene that encodes factor IX, a protein normally secreted into the blood by the liver, which functions as a component of the coagulation cascade. The clinical severity of hemophilia B is related to the amount of factor IX activity. Severe illnesses are associated with less than 1% of normal activity, while milder illnesses are associated with factor IX activity greater than 1% [Kaushansky, K. et al. & Williams, W.W. J. Williams hematology, (McGraw-Hill Medical, New York, 2010); Lofqvist, T. et al. , Et al. Journal of internal medicine 241,395-400 (1997)]. This suggests that even a small portion of editing therapy capable of restoring factor IX expression can have a significant effect on clinical outcomes by applying it to hepatocytes. Studies that modify a mouse model of hemophilia B using ZFNs shortly after birth have demonstrated that a modification of 3-7% was sufficient to reverse disease symptoms, and preclinical evidence for this hypothesis. [Li, H. et al. , Et al. Nature 475, 217-221 (2011)].

Disorders in which slight changes in gene product levels can affect clinical outcomes, and diseases in which edited cells have a fitness advantage, are therapeutically modified to allow high response rates given current technology. The threshold is low enough to be an ideal target for genome editing therapy. Targeting these diseases is now leading to successful editorial therapy at preclinical and Phase I clinical trials. Improvements in DSB repair pathway manipulation and nuclease delivery are needed to extend these promising outcomes to diseases with intermediate fitness predominance for edited cells or diseases that require large amounts of gene products for treatment. Become. The table below shows some examples of application of genome editing to therapeutic models, and the references in the table below and the references cited in those references are fully presented herein. Similarly incorporated herein by reference.

Using the CRISPR-Cas (eg Cas9) system, either correction of mutations by HDR through delivery systems, such as particle delivery systems, or insertion of modified gene sequences via HDR, as described herein advantageously. It is within the scope of one of ordinary skill in the art to target and respond to each of the pathological conditions in the above table from the present disclosure and knowledge in the art. Thus, certain embodiments include hemophilia B, SCID (eg, SCID-X1, ADA-SCID) or hereditary tyrosinemia mutation-bearing HSCs and hemophilia B, SCID (eg, SCID-X1, ADA). Includes contact with sgRNA- and -Cas (eg Cas9) protein-containing particles targeting the genomic loci of interest for -SCID) or hereditary tyrosinemia (eg, as in Li, Genovese or Yin). ). The particles may also contain an HDR template suitable for correcting the mutation; or the HSC may be contacted with a second particle or vector containing or delivering the HDR template. In this regard, it is mentioned that hemophilia B is an X-linked recessive inheritance disorder caused by a loss-of-function mutation in the gene encoding factor IX, an important component of the coagulation cascade. Restoring factor IX activity to more than 1% of that level in critically ill patients can transform the disease into a significantly milder form, with recombinant factor IX in such patients to achieve such levels. Prophylactic infusion from an early age generally seems to improve clinical complications. CRISPR- HSCs can be modified for hemophilia B using Cas (eg, Cas9) systems (eg, including suitable HDR templates that deliver the coding sequence of Factor IX); specifically, sgRNA is blood. Mutations that give rise to friendship B can be targeted, and HDR can provide coding for proper expression of factor IX. Mutant HSCs are contacted with sgRNAs that target mutant-and-Cas (eg, Cas9) protein-containing particles. The particles may also contain an HDR template suitable for correcting the mutation for proper expression of Factor IX; or the HSC is a second particle or vector containing or delivering the HDR template. May be in contact with. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier discussed herein.

Cartier, "Mini-Symposium: X-Linked Adrenoleukodystrophypa, Hematopoietic Stem -Linked Adrenoleukodystrophy) ”, Brain Pathology 20 (2010) 857-862 (incorporated herein by reference as all indicated, together with its references) using allogeneic hematopoietic stem cell transplantation (HSCT). There is recognition that normal lysosome enzymes have been delivered to the brains of patients with Harler's disease, and consideration of HSC gene therapy for the treatment of ALD. In two patients, peripheral CD34 + cells were collected after recruitment of granulocyte colony stimulating factor (G-CSF), myeloproliferative sarcoma virus enhancer, negative control region was deleted, and dl587rev primer binding site was replaced (dl587rev primer binding site). It was transduced with an MND) -ALD lentiviral vector. Patient-derived CD34 + cells were transduced with this MND-ALD vector at low concentrations for 16 hours in the presence of cytokines. Transduced CD34 + cells were frozen after transduction and various safety tests were performed on 5% of the cells, including specifically three replication competent trench virus (RCL) assays. The transduction efficacy of CD34 + cells ranged from 35% to 50%, with an average lentivirus-incorporated copy number of 0.65 to 0.70. After thawing of transduced CD34 + cells, patients were reinjected with more than 4.106 transduced CD34 + cells / kg, following complete bone marrow destruction by busulfan and cyclophosphamide. The patient's HSC was ablated to favor engraftment of the genetically modified HSC. Hematological recovery occurred on days 13-15 for the two patients. Almost complete immunological recovery occurred at 12 months for the first patient and at 9 months for the second patient. In contrast to using lentivirus, those skilled in the art, based on their knowledge in the art and the teachings of the present disclosure, target and modify mutations with respect to ALD in CRISPR-Cas (eg Cas9) systems (eg Cas9). HSCs can be modified using (including suitable HDR templates); specifically, sgRNA targets mutations in ABCD1 of genes located above the X chromosome encoding the peroxysome membrane transport protein ALD. And HDR can result in proper protein expression coding. The sgRNA that targets the mutation-and-Cas (eg Cas9) proteins containing the particles is contacted with an HSC having the mutation as in Cartier, such as CD34 + cells. The particles can also contain an HDR template suitable for modifying mutations to express the peroxisome membrane transport protein; or the HSC contains or delivers a second particle or vector that contains or delivers the HDR template. May be in contact with. Cells contacted in this way can optionally be treated as in Cartier. Cells contacted in this way can be administered as in Cartier.

WO 2015/148860 is cited, and through the teachings of this specification, the present invention includes methods and materials of these documents that apply in conjunction with the teachings of this specification. In certain aspects of blood-related disease gene therapy, methods and compositions for treating β-thalassemia may be adapted to the CRISPR-Cas system of the invention (see, eg, WO 2015/148860). In certain embodiments, WO 2015/148860 relates to the treatment or prevention of β-thalassemia, or its symptoms, eg, by altering the gene for B cell CLL / lymphoma 11A (BCL11A). The BCL11A gene is also known as B cell CLL / lymphoma 11A, BCL11A-L, BCL11A-S, BCL11AXL, CTIP1, HBFQTL5 and ZNF. BCL11A encodes a zinc finger protein involved in the regulation of globin gene expression. Altering the BCL11A gene (eg, one or both alleles of the BCL11A gene) can increase γ-globin levels. γ-globin can replace β-globin in the hemoglobin complex to effectively transport oxygen to tissues, which can improve the β-thalassemia disease phenotype.

Pamphlets of WO 2015/148863 are also mentioned, and through the teachings of this specification, the present invention includes the methods and materials of these documents that can be applied to the CRISPR-Cas system of the present invention. In some embodiments of the treatment and prevention of the hereditary blood disease sickle cell disease, WO 2015/148863 pamphlet comprises altering the BCL11A gene. Altering the BCL11A gene (eg, one or both alleles of the BCL11A gene) can increase γ-globin levels. γ-globin can replace β-globin in the hemoglobin complex to effectively transport oxygen to tissues, which can improve the sickle cell disease phenotype.

In certain aspects of the invention, by adapting the CRISPR-Cas system of the invention, methods and compositions involving editing of the target nucleic acid sequence, or regulation of expression of the target nucleic acid sequence, and its application in connection with cancer immunotherapy. Is included. Because one or more changes in one or more T cell expression genes, such as FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC and / or TRBC genes, affect T cell proliferation, survival and / or function. See the application of gene therapy in Pamphlet International Publication No. 2015/161276 with methods and compositions that can be used in. In a related embodiment, T cell proliferation is associated with one or more T cell expression genes, such as the CBLB and / or PTPN6 gene, FAS and / or BID gene, CTLA4 and / or PDCDI and / or TRAC and / or TRBC gene. Can be affected by change.

Chimeric antigen receptor (CAR) 19 T cells exhibit anti-leukemic effects in malignant lesions of patients. However, leukemia patients often do not have enough T cells to collect, which means that treatment should include modified T cells from the donor. Therefore, building a donor T cell bank is beneficial. Qasim et al. ("First Clinical Application of Talen Engineered Universal CAR19 T Cells in B-ALL", ASH 57th Ann. 2015, Abstract 2046 (https://ash.confex.com/ash/2015/webprogram/Paper81653.html online publication November 2015) eliminates graft-versus-host disease risk by disrupting T cell receptor expression and CD52 targeting In addition, CD52 cells are targeted to be insensitive to alemtuzumab, which in turn is host-mediated by human leukocyte antigen (HLA) mismatch CAR19 T cells. It became possible to prevent rejection. Researchers used a 3rd generation self-inactivating lentivirus vector encoding 4g7 CAR19 (CD19 scFv-4-1BB-CD3ζ) linked to RQR8, followed by Two pairs of TALEN mRNAs for multiple targeting to both the T cell receptor (TCR) α constant chain locus and the CD52 locus were electroperforated into the cells. Depletion using / βTCR depletion resulted in <1% TCR expressing T cell product (UCART19), of which 85% expressed CAR19 and 64% became CD52 negative. Of this modified CAR19 T cell. Administration treated a patient with recurrent acute lymphoblastic leukemia. The teachings provided herein are, but are not limited to, blood myeloid and lymphoid cells such as T cells, B cells, simply. Modified hematopoietic stem cells and their progeny, including spheres, macrophages, neutrophils, basal spheres, eosinophils, erythrocytes, dendritic cells, and macronuclear or platelets, and natural killer cells and their precursors and ancestors. Provided is an effective method of providing such cells, which can be modified by knockout, knockin, or otherwise regulation of the target, eg, described above. CD52 and other targets as described, such as CXCR4 and PD-1, can be removed or regulated without limitation. Thus, the compositions, cells, and methods of the invention, along with alterations in the administration of T cells or other cells to a patient, regulate and, without limitation, the immune response of malignant lesions, viral infections, and immune disorders. It can be used for treatment.

WO 2015/148670 Pamphlet is mentioned, and through the teachings of this specification, the present invention includes the methods and materials of this document that apply in conjunction with the teachings of this specification. In certain aspects of gene therapy, methods and compositions for editing target sequences associated with or associated with the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) are contemplated. In a related aspect, the inventions described herein include the prevention and treatment of HIV infection and AIDS by introducing one or more mutations into the gene for CC chemokine receptor type 5 (CCR5). To do. The CCR5 gene is also known as CKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22, and CC-CKR-5. In a further aspect, the inventions described herein provide prevention or reduction of HIV infection and / or prevention or reduction of the ability of HIV to invade host cells, eg, in a subject already infected. Include. Illustrative host cells for HIV include, but are not limited to, CD4 cells, T cells, gut-associated lymphoid tissue (GALT), macrophages, dendritic cells, bone marrow progenitor cells, and microglia. Viral invasion into host cells requires interaction of the viral glycoproteins gp41 and gp120 with both CD4 and co-receptors, such as CCR5. If a co-receptor, such as CCR5, is not present on the surface of the host cell, the virus cannot bind to and invade the host cell. Therefore, the progression of the disease is hindered. Invasion of the HIV virus into the host cell is prevented by knocking out the CCR5 of the host cell or by knocking down, for example, by introducing a protective mutation (such as the CCR5 delta 32 mutation).

X-linked chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to lack or decreased activity of phagocytic NADPH oxidase. Use a CRISPR-Cas (Cas9) system that targets and corrects mutations (lack or decreased activity of phagocytic NADPH oxidase), including, for example, a suitable HDR template that delivers the coding sequence of phagocytic NADPH oxidase; Specifically, sgRNA can target mutations that give rise to CGD (deficiency of phagocytic NADPH oxidase), and HDR can provide coding for appropriate phagocytic NADPH oxidase expression. The mutated HSC is contacted with an sgRNA that targets the mutant-and-Cas (Cas9) protein-containing particles. The particles may also contain an HDR template suitable for modifying the mutation for proper expression of phagocytic NADPH oxidase; or the HSC may contain or deliver a second particle or the HDR template. It may be in contact with the vector. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier.

Fanconi anemia: At least 15 genes (FANCA, FANCB, FANCC, FANCD1 / BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCLJ / BACH1 / BRIP1, FANCL / PHF9 / POG, FANCM, FANCL / PALB2, FA Mutations in Rad51C and FANCP / SLX4 / BTBD12) can cause Fanconi anemia. The proteins produced by these genes are involved in a cellular process known as the FA pathway. The FA pathway is turned on (activated) when the process of making a new copy of DNA, called DNA replication, is blocked due to DNA damage. The FA pathway pumps a particular protein into the area of injury, which triggers DNA repair to begin, thus allowing DNA replication to continue. The FA pathway is particularly responsive to certain types of DNA damage known as interstrand crosslinks (ICLs). ICL occurs when two DNA components (nucleotides) on the reverse strand of DNA are abnormally bound or linked together, thereby terminating the process of DNA replication. ICL can be caused by the accumulation of toxic substances produced in the body or by treatment with certain cancer therapeutics. Eight proteins associated with Fanconi anemia combine into a group to form a complex known as the FA core complex. The FA core complex activates two proteins called FANCD2 and FANCI. Activation of these two proteins brings the DNA repair protein into the ICL range, thus removing the crosslinks and allowing DNA replication to continue. FA core complex. More specifically, the FA core complex is a nuclear multiprotein complex consisting of FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functions as an E3 ubiquitin ligase, and is composed of FANCD2 and FANCI. Mediates the activation of the ID complex, which is a heterodimer. When the FA core complex is monoubiquitinated, it interacts with classical tumor suppressors downstream of the FA pathway, including FANCD1 / BRCA2, FANCN / PALB2, FANCJ / BRIP1, and FANCO / Rad51C, thereby homologous recombination. Contributes to DNA repair by (HR). 80-90% of FA cases result from mutations in one of three genes, FANCA, FANCC, and FANCG. These genes provide instructions for the production of components of the FA core complex. Mutations in such genes associated with the FA core complex can render the complex non-functional and disrupt the entire FA pathway. As a result, DNA damage is not repaired efficiently and ICL accumulates over time. Geiselhart, "Review Article, Disruption of Signaling Through the Fanconi Anemia Pathway Leads to Dysfunctional Hematopoietic Stem Cell Biology: Review Article, Disrupted Signaling Through The Fantasy Ane to Dysfunctional Hematopoietic Stem Cell Biology: Underlying Mechanisms and Potential Therapeutic Strategies) ”, Anemia Volume 2012 (2012), Anemia Volume 2012 (2012) doi. In org / 10.1155 / 2012/265790, animal experiments involving FA and intrafemoral injection of a lentivirus encoding the FANCC gene resulting in modification of HSC in vivo were considered. A CRISPR-Cas (Cas9) system that targets one or more mutations associated with FA, such as one or more mutations in FANCA, FANCC, or FANCG that give rise to FA, and FANCA, FANCC, or Use a CRISPR-Cas (Cas9) system, each with one or more sgRNAs and one or more HDR templates that provide one or more modified expressions of FANCG; for example, sgRNAs target mutations for FANCC. And HDR can provide coding for proper expression of FANCC. One associated with FA, such as one or more mutations (eg, one or more mutations for any one or more of FANCA, FANCC or FANCG) to an HSC having one or more mutations. The above) targeting sgRNA- and-Cas (Cas9) protein-containing particles are brought into contact with each other. The particles also include one or more HDR templates suitable for correcting mutations for proper expression of one or more proteins involved in FA, such as any one or more of FANCA, FANCC or FANCG. It may be contained; or the HSC may be contacted with a second particle or vector containing or delivering the HDR template. Cells contacted in this way can be administered; and optionally processed / expanded; see Cartier.

With respect to the particles discussed herein (eg, one or more sgRNAs and Cas (Cas9), optionally one or more HDR templates, or those containing one or more HDR templates; eg, Hematopoietic disease B, SCID, SCID-X1, ADA-SCID, hereditary tyrosineemia, β-thalassemia, X-chain CGD, Viscott-Oldrich syndrome, fancony anemia, adrenoleukodystrophy (ALD), heterozygous White dystrophy (MLD), HIV / AIDS, immunodeficiency disorders, hematological conditions, or genetic lithosome storage disease) are advantageous for one or more sgRNA and Cas (Cas9) protein mixtures (optionally). Such a mixture containing one or more HDR templates, or only containing one or more HDR templates if separate particles are desired for one or more templates), It is obtained or can be obtained by mixing with a mixture containing or consisting essentially of or consisting of phospholipids, biodegradable polymers, lipoproteins and alcohols (where one or more sgRNAs are). Target one or more loci of HSC).

In fact, the present invention is particularly suitable for the treatment of genetic hematopoietic disorders by genome editing and for the treatment of immunodeficiency disorders such as genetic immunodeficiency disorders, particularly by using the particle techniques discussed herein. Genetic immunodeficiency is a disease in which the genome editing interventions of the present invention can be successful. The reason is that hematopoietic cells, of which immune cells are a subset, are therapeutically accessible. Hematopoietic cells can be removed from the body for autologous or allogeneic transplantation. In addition, certain genetic immunodeficiencies, such as severe combined immunodeficiency (SCID), result in a proliferative disadvantage for immune cells. A collection of genetic lesions that cause SCID by rare spontaneous "return" mutations shows that even a modification of a single lymphocyte ancestor can be sufficient to restore a patient's immune function. .. .. /. .. /. .. / Users / t_kowarski / AppData / Local / Microsoft / Windows / Temporary Internet Files / Content. Outlook / GA8VY8LK / Treating SCID for Ellen. docx-_ENREF_1. Bousso, P.M. , Et al. "Diversity, functionality, and stability of the T cell repertoire developed in vivo cell cell singing, diversity, functionality, and stability of the T cell repertoire derived from a single human T cell precursor in vivo. ". See Proceedings of the National Academia of Sciences of the United States of America 97, 274-278 (2000). The selective dominance of edited cells allows even low levels of editing to produce therapeutic effects. This effect of the present invention has a negative effect on fitness of erythroid progenitor cells due to hemoglobin deficiency, including SCID, Viscott-Oldrich syndrome, and other genetic hematopoietic disorders such as α- and β-thalassemia. It can be found in other pathologies referred to herein.

NHEJ and HDR DSB repair activity varies greatly depending on cell type and cell status. NHEJ is not highly regulated by the cell cycle and is efficient across all cell types, allowing high levels of gene disruption in contactable target cell populations. In contrast, HDR works primarily during the S / G2 phase and is therefore restricted to actively dividing cells, and treatments that require accurate genomic modification are limited to mitotic cells [Ciccia, A. .. & Elledge, S.A. J. Molecular cell 40, 179-204 (2010); Chapman, J. Mol. R. , Et al. Molecular cell 47, 497-510 (2012)].

HDR-mediated modification efficiency can be controlled by the metamorphic state or sequence of the target locus, or the specific repair template configuration used (single-to-double, long-to-short homology arm) [Hacein- Bey-Abina, S.A. , Et al. The New England journal of medicine 346, 1185-1193 (2002); Gaspar, H. et al. B. , Et al. The Lancet 364,2181-2187 (2004); Beamer, K. et al. J. , Et al. G3 (2013)]. The relative activity of NHEJ and HDR mechanisms in target cells can also affect gene modification efficiency because these pathways can compete for DSB elimination [Beumer, K. et al. J. , Et al. Proceedings of the National Academia of Sciences of the United States of America 105, 19821-19826 (2008)]. HDR also presents delivery challenges not found in NHEJ strategies, as it requires simultaneous delivery of nucleases and repair templates. In fact, these constraints have so far led to low levels of HDR in therapeutically relevant cell types. Therefore, clinical interpretation focuses primarily on NHEJ strategies for the treatment of disease, however, proof-of-concept preclinical HDR treatments are currently reported for mouse models of hemophilia B and hereditary tyrosinemia [Li, H. , Et al. Nature 475, 217-221 (2011); Yin, H. et al. , Et al. Nature biotechnology 32,551-553 (2014)].

Delivery of these multiple parts poses a substantial challenge compared to small molecule therapeutics, as any given genome editing application can include a combination of proteins, small RNA molecules, and / or repair templates. Two major strategies have been developed for the delivery of genome editing tools: ex vivo and in vivo. In exobibo treatment, affected cells are removed from the body, edited, and then transplanted back into the patient. Exobibo editing has the advantage that the target cell population is well defined and it is possible to identify specific dosages of therapeutic molecules delivered to the cells. The latter consideration can be particularly important when off-target modification is a concern, as such mutations can be reduced by titrating the amount of nuclease (Hsu et al., 2013). Another advantage of the Exobibo approach is that efficient delivery systems of proteins and nucleic acids to cultured cells have been developed for research and gene therapy applications, which can typically achieve high edit rates. ..

The Exobibo approach may have the drawback of limiting its application to a small number of diseases. For example, the target cell must have the ability to survive in vitro manipulation. For many tissues, such as the brain, culturing cells in vitro is a major challenge because they either cannot survive or lose the properties required for their function in vivo. Therefore, in view of the present disclosure and knowledge in the art, CRISPR-Cas (Cas9) -based exovivo therapy is possible for tissues with adult stem cell populations suitable for exovivo culture and manipulation, such as hematopoietic systems. [Bunn, H. et al. F. & Aster, J.M. Pathophysiology of blood disorders, (McGraw-Hill, New York, 2011)].

In vivo genome editing involves directing the delivery of the editing system to cell types in its natural tissue. In vivo editing allows the treatment of diseases in which the affected cell population is unsuitable for ex vivo manipulation. In addition, the nuclease is delivered to cells in situ, allowing treatment of multiple tissues and cell types. Perhaps due to these properties, in vivo treatment is applicable to a wider range of diseases compared to ex vivo therapy.

To date, in vivo editing has generally been achieved by the use of viral vectors with defined tissue-specific tropism. Such vectors are currently limited in terms of cargo carrying capacity and tropism, and this treatment is limited to organ systems such as the liver, muscle and eye where transduction with clinically useful vectors is efficient. [Kotterman, M. et al. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Nguyen, T. et al. H. & Ferry, N.M. Gene therapy 11 Suppl 1, S76-84 (2004); Boye, S. et al. E. , Et al. Molecular therapy: the journal of the American Society of Gene Therapy 21,509-519 (2013)].

A potential barrier to in vivo delivery is an immune response that can occur in response to large amounts of virus required for treatment, but this phenomenon is not unique to genome editing and is also found in other virus-based gene therapies. [Bessis, N. et al. , Et al. Gene therapy 11 Suppl 1, S10-17 (2004)]. Also, peptides of the editing nuclease itself may be presented on MHC class I molecules and stimulate the immune response, but at the preclinical level there is little evidence to support this. Another major challenge for this therapy is controlling the in vivo distribution, and thus the dosage of genome editing nucleases, resulting in off-target mutation profiles that can be difficult to predict. However, in the context of this disclosure and knowledge in the art, including the use of virus-based and particle-based therapies used in the treatment of cancer, in vivo modifications of HSCs (eg, by delivery by either particles or viruses). Is within the scope of those skilled in the art.

Exovivo Editing Therapy: Due to many years of clinical views on hematopoietic cell purification, culture and transplantation, diseases affecting the blood system such as SCID, Fanconi anemia, Viscott Oldrich syndrome and sickle cell anemia are associated with Exovivo editing therapy. It has become the main focus. Another reason for the focus on hematopoietic cells is that relatively efficient delivery systems already exist, thanks to previous efforts to design gene therapies for hematopoietic disorders. Due to these advantages, this treatment can be applied to diseases in which the editorial cells have a fitness advantage and therefore a small number of engrafted editorial cells can expand to treat the disease. One such disease is HIV, where infection results in a fitness disadvantage for CD4 + T cells.

In recent years, exobibo editing therapies have been extended to include gene modification strategies. The barrier to HDR in exovivo has been overcome in a recent paper by Genovese and co-workers, who found the mutant IL2RG gene in hematopoietic stem cells (HSCs) from patients with SCID-X1. [Geneovese, P. et al. , Et al. Nature 510, 235-240 (2014)]. Genovese et. al. Achieved genetic modification in HSC using a multidisciplinary strategy. First, HSC was transduced using an integrated defective wrench virus containing an HDR template encoding the therapeutic cDNA of IL2RG. After transduction, cells were electroporated with ZFN-encoding mRNAs that target mutant hotspots in IL2RG and stimulate HDR-based gene modification. In order to increase the HDR rate, the culture conditions were optimized for small molecules to promote HSC division. With optimized culture conditions, nucleases and HDR templates, HSCs with modified genes from SCID-X1 patients were obtained in culture at therapeutically significant rates. HSCs from non-affected individuals who underwent the same genetic modification procedure were able to maintain long-term hematopoiesis, the gold standard for HSC function, in mice. HSCs have the ability to give rise to all hematopoietic cell types and can be autologously transplanted, making HSCs a highly useful cell population for all hematopoietic genetic disorders [Weissman, I. et al. L. & Shizuru, J.M. A. Blood 112,3543-3553 (2008)]. Genetically modified HSCs can, in principle, be used to treat a wide range of genetic blood disorders, making this trial an exciting breakthrough in therapeutic genome editing.

In vivo editing therapy: From the present disclosure and knowledge in the art, in vivo editing can be advantageously used. For organ systems with efficient delivery, there are already a number of exciting preclinical treatment successes. The first successful cases of in vivo editing therapy were demonstrated in a mouse model of hemophilia B [Li, H. et al. , Et al. Nature 475, 217-221 (2011)]. As mentioned earlier, hemophilia B is an X-linked recessive genetic disorder caused by a loss-of-function mutation in the gene encoding factor IX, an important component of the coagulation cascade. When factor IX activity recovers above 1% of that level in critically ill patients, the disease can be significantly transformed into a mild form, which prophylactically recombined factor IX in such patients from an early age. Achieving such levels by injecting into is generally expected to improve clinical complications [Lofqvist, T. et al. , Et al. Journal of internal medicine 241,395-400 (1997)]. Therefore, low levels of HDR gene modification are sufficient to alter the clinical outcome of a patient. In addition, Factor IX is synthesized and secreted by the liver, which is an organ that can be efficiently transduced by viral vectors encoding editorial systems.

Using the hepatic adeno-associated virus (AAV) serotype encoding the ZFN and modified HDR templates, up to 7% of the mutant humanized Factor IX gene was modified in mouse liver [Li, H]. .. , Et al. Nature 475, 217-221 (2011)]. This improved the clot formation dynamics, which is a measure of coagulation cascade function, demonstrating for the first time that in vivo editing therapy is not only feasible but also effective. As discussed herein, those skilled in the art will be able to target mutations in X-linked recessive inheritance to restore loss-of-function mutations from the teachings and knowledge of the art, such as Li. The HDR template and CRISPR-Cas (Cas9) system contained in are in a situation where hemophilia B can be treated.

Based on this study, other groups have recently created mutations that provide treatment of mouse models of hereditary tyrosinemia and protection from cardiovascular disease using in vivo genome editing of the liver with CRISPR-Cas. Has been successful. These two different applications demonstrate the versatility of this approach for disorders associated with liver dysfunction [Yin, H. et al. , Et al. Nature biotechnology 32,551-553 (2014); Ding, Q. et al. , Et al. Circulation research 115, 488-492 (2014)]. In vivo editing needs to be applied to other organ systems to prove that this strategy is widely applicable. Efforts are currently underway to optimize both viral and non-viral vectors to broaden the range of disorders that can be treated with this treatment [Kotterman, M. et al. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Yin, H. et al. , Et al. Nature reviews. Genetics 15,541-555 (2014)]. As discussed herein, one of ordinary skill in the art inherits from the teachings of the specification and knowledge in the art, such as Yin, in HDR templates and CRISPR-Cas (Cas9) systems containing mutation-targeting particles. You are in a position to deal with sexual tyrosinemia.

Targeted deletion, therapeutic application: Targeted deletion of genes may be preferred. Thus, immunodeficiency disorders, hematological conditions, or genetic lysosome storage disorders, such as hemophilia B, SCID, SCID-X1, ADA-SCID, hereditary tyrosineemia, β-salasemia, X-linked CGD, vi. Scott Oldrich Syndrome, Funcony Anemia, Adrenoleukodystrophy (ALD), Severe Combined Immunodeficiency Dystrophy (MLD), HIV / AIDS, Genes Involved in Other Metabolic Disorders, Genes Encoding Misfolding Proteins Related to Diseases, Genes that lead to disease-related loss of function; in general, mutations that can be targeted in HSCs using any delivery system discussed herein in a particle system that is considered advantageous are preferred.

In the present invention, in particular, the immunogenicity of the CRISPR enzyme was initially shown in Tangri et al in relation to erythropoietin and can be reduced according to subsequently developed techniques. Therefore, directed evolution or rational design can be used to reduce the immunogenicity of the CRISPR enzyme (eg, Ca9) in the host species (human or other species).

Genome Editing: Using the CRISPR / Cas (Cas9) system of the present invention, including those discussed herein (see also WO 2013163628), with TALENs and ZFNs and lentiviruses. Attempts have been made, but success has been limited. Gene mutations can be corrected.

Adoptive Cell Therapy The present invention also contemplates the use of the CRISPR-Cas system described herein, eg, the Cas9 effector protein system, for modifying cells for adoptive therapy. Aspects of the present invention include the adoptive transfer of immune system cells such as T cells specific for a particular antigen, eg, a tumor-related antigen (Maus et al., 2014, “Adaptive Immunotherapy for Cancer or Virus”. Cancer or Viruses) ”, Annual Review of Immunotherapy, Vol. 32: 189-225; Rosenberg and Restifo, 2015,“ Adoptive cell transfer as an individualized immunotherapy for human cancer. , Science Vol. 348 no. 6230 pp. 62-68; Restifo et al., 2015, "Adaptive immunotherapy for cancer: harnessing the T cell. Immunol. 12 (4): 269-281; and Jenson and Riddell, 2014, "Design and Implementation of immunotherapy with immunotherapy with chimeric immunotherapy". (See Immunol Rev. 257 (1): 127-144). T cells are genetically modified using various strategies, eg, by altering the specificity of the T cell receptor (TCR), eg, by introducing novel TCR α and β chains with selected peptide specificity. Obtain (US Pat. No. 8,697,854; PCT Patent Publication: International Publication No. 2003020763, International Publication No. 2004033685, International Publication No. 2004040044, International Publication No. 20050114215, International Publication No. 2006008830 Pamphlet, International Publication No. 2008038002 Pamphlet, International Publication No. 2008039818 Pamphlet, International Publication No. 2004074322 Pamphlet, International Publication No. 200511595 Pamphlet, International Publication No. 20012625962 Pamphlet, International Publication No. 2013166321 Pamphlet, International Publication No. 2013039889 Pamphlet, International Publication No. 2014088663, International Publication No. 2014083173; see US Pat. No. 8,088,379).

Chimeric antigen receptors (CARs) may be used in place of or in addition to TCR modifications to create immune-responsive cells such as T cells that are specific for selective targets such as malignant cells and are diverse. Receptor chimeric constructs are described (US Pat. Nos. 5,843,728; 5,851,828; 5,912,170; 6,004). 811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 6,753,162; 8, 211,422; and WO 9215322). Alternative CAR constructs can be characterized as belonging to a continuous generation. The primary CAR typically consists of a single chain variable fragment of an antibody specific for an antigen, eg, a mobile linker to the transmembrane and intracellular signaling domain of either CD3ζ or FcRγ, eg CD8α. Includes _VL linked to _{VH of} a specific antibody, linked by a hinge domain and a CD8α transmembrane domain (scFv-CD3ζ or scFv-FcRγ; US Pat. No. 7,741,465; US Pat. , 912,172; see US Pat. No. 5,906,936). The second generation CAR incorporates the intracellular domain of one or more costimulatory molecules such as CD28, OX40 (CD134), or 4-1BB (CD137) into the endodomain (eg scFv-CD28 / OX40 / 4-1BB). -CD3ζ; US Pat. No. 8,911,993; US Pat. No. 8,916,381; No. 8,975,071; No. 9,101,584; No. 9,102,760; see 9,102,761). The third generation CAR comprises a combination of co-stimulating end domains such as CD3ζ-chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domain (eg,). scFv-CD28-4-1BB-CD3ζ or scFv-CD28-OX40-CD3ζ; US Pat. No. 8,906,682; US Pat. No. 8,399,645; US Pat. No. 5,686,281; Specification; See International Publication No. 2014134165; International Publication No. 2012079000). Alternatively, co-stimulation can be achieved, for example, by expressing CAR in antigen-specific T cells selected to be activated and expanded after association of their native αβTCR by an antigen on a professional antigen-presenting cell with co-stimulation. It may be planned. In addition, additional engineered receptors may be provided on immune-responsive cells, eg, which enhances targeting of T cell attacks and / or minimizes side effects.

Alternative techniques such as protoplast fusion, lipofection, transfection or electroporation may be used to transform target immunoresponsive cells. A wide variety of vectors can be used, including retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, plasmids or transposons such as Sleeping Beauty transposon (US Pat. No. 6,489,458; The same No. 7,148,203; the same No. 7,160,682; the same No. 7,985,739; the same No. 8,227,432), using it. CAR can be introduced using, for example, a second-generation antigen-specific CAR that signals through either CD3ζ and CD28 or CD137. Viral vectors include, for example, HIV, SV40, EBV, HSV or BPV based vectors.

Cells targeted for transformation include, for example, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor infiltrating lymphocytes ( TIL) or pluripotent stem cells from which lymphoid cells can differentiate can be mentioned. T cells expressing the desired CAR may be selected, for example, by γ-irradiated activation and co-culture with proliferating cells (AaPC) (co-expressing cancer antigens and co-stimulatory molecules). The engineered CAR T cells may be expanded by co-culturing on AaPC in the presence of soluble factors such as IL-2 and IL-21. This expansion may be performed, for example, to provide memory CAR + T cells (which may be assayed, for example, by non-enzymatic digital arrays and / or multipanel flow cytometry). In this way, CAR T cells with cytotoxic activity specific for antigen-carrying tumors can be provided (optionally with the production of desired chemokines such as interferon-γ). This type of CAR T cell can be used, for example, for the treatment of tumor xenografts, eg, in animal models.

Techniques such as those described above treat diseases such as neoplasms and / or survive subjects with them, eg, by administering an effective amount of immune-responsive cells containing an antigen-recognizing receptor that binds to the antigen of choice. Can be adapted to provide a method of increasing, where binding activates immune-responsive cells, thereby causing disease (such as neoplasms, pathogen infections, autoimmune disorders, or allograft reactions). To treat or prevent. Dose determination of CAR T cell therapy may involve administration of, for example, 106-109 cells / kg with or without a process of lymphocyte depletion, eg, with cyclophosphamide.

In one embodiment, the treatment can be administered to a patient undergoing immunosuppressive treatment. A cell or cell population can become resistant to at least one immunosuppressive drug due to the inactivation of the gene encoding the receptor for such immunosuppressive drug. Without being bound by theory, immunosuppressive therapy should aid in the selection and expansion of immunoresponsive cells or T cells according to the invention in the patient's body.

Administration of cells or cell populations according to the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The cells or cell population may be administered to the patient subcutaneously, intradermally, intratumorally, intranodesally, intramedullarily, intramuscularly, intravenously or intralymphally, or intraperitoneally. In one embodiment, the cell composition of the invention is preferably administered by intravenous injection.

Administration of cells or cell populations consists of administration of 10 ⁴ to ⁹ cells per kg body weight, preferably 10 ⁵ to 1 to ⁶ cells / kg body weight (including all integer cell numbers within these ranges). obtain. Dose determination in CAR T cell therapy can involve, for example, administration of 10 ⁶ to ⁹ cells / kg with or without the process of lymphocyte depletion by, for example, cyclophosphamide. The cells or cell population can be administered at one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as two or more doses over a period of time. The timing of administration is within the judgment of the managing physician and depends on the clinical condition of the patient. The cells or cell populations can be obtained from any source, such as blood banks or donors. Although individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount means an amount that provides therapeutic or prophylactic benefit. The dosage administered may depend on the age, health and weight of the recipient, in some cases the type of combination treatment, the frequency of treatment and the nature of the desired effect.

In another embodiment, an effective amount of cells or a composition comprising those cells is administered parenterally. The administration may be intravenous administration. Administration may be direct by injection into the tumor.

To prevent possible adverse reactions, engineered immunoresponsive cells may be equipped with transgenic safety switches in the form of transgenes that make the cells vulnerable to exposure to specific signals. The simple herpesvirus thymidine kinase (TK) gene can be used in this way, for example, by introduction into allogeneic T lymphocytes used for donor lymphocyte injection after stem cell transplantation (Greco, et al., "TK suicide". Improving the safety of cell therapy with genes (Improving the safety of cell therapy with the TK-thymidine gene) ". Front. Pharmacol. 2015; 6:95). In such cells, administration of a nucleoside prodrug such as ganciclovir or acyclovir causes cell death. Alternative safety switch constructs include, for example, inducible caspase-9 induced by administration of a small molecule dimerizing agent that combines two non-functional icasp9 molecules to form an active enzyme. A wide variety of alternative methods for controlling cell proliferation are described (US Patent Application Publication No. 20130071414; International Publication No. 2011146862; International Publication No. 2014011987; International Publication No. 2013040371. Zhou et al. BLOOD, 2014, 123/25: 3895-3905; Di Stasi et al., The New England Journal of Medicine 2011; 365: 1673-1683; Sadalein M, The New 1735-173; Ramos et al., Stem Cells 28 (6): 1107-15 (2010)).

Further improvements in adoptive therapy may use genome editing to tailor immunoresponsive cells to an alternative implementation method, eg, to provide edited CAR T cells (Poirot et al., 2015, " "Off-the-shelf" T-cell production platform by multiple genome editing of adopted child T-cell immunotherapy (Multiplex genome-edited T-cell manufacturing plateform for "off-the-self" adaptive T-cell Mexico 18): See 3853). Cells can be edited using any CRISPR system and method of use thereof as described herein. The CRISPR system can be delivered to immune cells by any of the methods described herein. In a preferred embodiment, cells are edited with exobibo and transferred to the subject in need. Immune-responsive cells, CAR T cells or any cells used for adoption can be edited. Editing eliminates potential alloreactive T cell receptors (TCRs), disrupts chemotherapeutic targets, blocks immune checkpoints, activates T cells, and / or is functionally depleted. It may be performed to increase the differentiation and / or proliferation of dysfunctional or dysfunctional CD8 + T cells (PCT Patent Publication: International Publication No. 2013176915, International Publication No. 2014059173, International Publication No. 2014172606, See International Publication No. 2014184744 and International Publication No. 2014191128). Editing can result in gene inactivation.

Inactivating a gene is intended to mean that the gene of interest is not expressed in functional protein form. In a detailed embodiment, the CRISPR system specifically catalyzes cleavage in one target gene, thereby inactivating the target gene. The induced cleavage of nucleic acid chains is generally repaired by a specific mechanism of homologous recombination or non-homologous end binding (NHEJ). However, NHEJ is an incomplete repair process that often causes changes in the DNA sequence at the cleavage site. Repair by non-homologous end joining (NHEJ) often results in small insertions or deletions (indels) and can be used to create specific gene knockouts. Cells in which a cleavage-induced mutagenesis event has occurred can be identified and / or selected by methods well known in the art.

The T cell receptor (TCR) is a cell surface receptor involved in the activation of T cells in response to antigen presentation. TCR is generally composed of two strands, α and β, which assemble to form a heterodimer and associate with the CD3 transduction subunit to form a T cell receptor complex present on the cell surface. Form. Each α and β chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and stationary (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. For immunoglobulin molecules, the variable regions of the α and β chains are generated by V (D) J recombination, creating a highly antigen-specific diversity within the T cell population. However, in contrast to immunoglobulins that recognize intact antigens, T cells are activated by processed peptide fragments associated with MHC molecules, introducing an extra aspect of antigen recognition by T cells known as MHC constraints. To do. Recognition of MHC differences between donors and recipients via the T cell receptor leads to T cell proliferation and the potential development of graft-versus-host disease (GVHD). Inactivating TCRα or TCRβ results in the removal of TCR from the surface of T cells, thereby preventing allogeneic antigen recognition and thus GVHD. However, disruption of the TCR generally results in the removal of CD3 signaling components, altering the means of further T cell expansion.

Allogeneic cells are rapidly rejected by the host immune system. Allogeneic leukocytes present in non-irradiated blood products have been demonstrated to persist for up to 5-6 days (Boni, Muranski et al. 2008 Blood 1; 112 (12): 4746-54). Therefore, to prevent rejection of allogeneic cells, the host's immune system usually must be suppressed to some extent. However, in the case of adoptive cell transfer, the use of immunosuppressive drugs also has adverse effects on the introduced therapeutic T cells. Therefore, in order to effectively use adoptive immunotherapy techniques under these conditions, the introduced cells may need to be resistant to immunosuppressive therapy. Thus, in a detailed embodiment, the present invention preferably inactivates at least one gene that edits an immunosuppressant target so that the T cell is resistant to the immunosuppressant. It further includes a step of modification. Immunosuppressants are agents that suppress immune function by one of several mechanisms of action. Immunosuppressants are, but are not limited to, calcineurin inhibitors, rapamycin targets, interleukin-2 receptor α-chain blockers, inosin monophosphate dehydrogenase inhibitors, dihydrofolate reductase inhibitors, corticosteroids or immunosuppressive metabolic antagonists. Can be. The present invention makes it possible to impart immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in the T cells. As a non-limiting example, the target of an immunosuppressive agent can be a receptor for an immunosuppressive agent, such as CD52, a glucocorticoid receptor (GR), an FKBP family gene member and a cyclophilin family gene member.

Immune checkpoints are inhibitory pathways that slow or arrest the immune response and prevent excessive tissue damage from uncontrolled immune cell activity. In certain embodiments, the targeted immune checkpoint is the programmed death-1 (PD-1 or CD279) gene (PDCD1). In other embodiments, the targeted immune checkpoint is a cytotoxic T lymphocyte-related antigen (CTLA-4). In a further embodiment, the targeted immune checkpoint is another member of the CD28 and CTLA4 Ig superfamily, such as BTLA, LAG3, ICOS, PDL1 or KIR. In yet another embodiment, the targeted immune checkpoint is a member of the TNFR superfamily, such as CD40, OX40, CD137, GITR, CD27 or TIM-x3.

Further immune checkpoints include the Src homology 2-domain-containing protein tyrosine phosphatase 1 (SHP-1) (Watson HA, et al., "SHP-1: Next Checkpoint Target for Cancer Immunotherapy?" (SHP-1: the next checkpoint tyrosine for cancer immunotherapy?) ”Biochem Soc Trans. 2016 Apr 15; 44 (2): 356-62). SHP-1 is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T cells, it is a negative regulator of antigen-dependent activation and proliferation. Although it is a cytoplasmic protein and therefore unsuitable for antibody-mediated therapy, its role in activation and proliferation makes SHP-1 attractive for genetic manipulation in adoption strategies, such as chimeric antigen receptor (CAR) T cells. Target. Immune checkpoints may also include T cell immune receptors, including Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9) and VISTA (Le Mercier I, et al., (2015) "Beyond PD-1". , Gen Z of negative checkpoint regulators (Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint receptors) ". Front. Immunel. 6: 418).

WO 2014172606 increases the proliferation and / or activity of depleted CD8 + T cells and reduces CD8 + T cell depletion (eg, reduces functionally depleted or non-responsive CD8 + immune cells). With respect to the use of MT1 and / or MT1 inhibitors for. In certain embodiments, metallothionein is targeted by gene editing in adopted T cells.

In certain embodiments, the target of gene editing can be at least one target locus involved in the expression of immune checkpoint proteins. Such targets include, but are not limited to, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9. , CD244 (2B4), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10 IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40, CD137, GITR, CD27, Can be done. In a preferred embodiment, the loci involved in the expression of the PD-1 or CTLA-4 gene are targeted. In other preferred embodiments, gene combinations such as, but not limited to, PD-1 and TIGIT are targeted.

In other embodiments, at least two genes are edited. The pair of genes is not limited, but is limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα. , BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRα, SIGLEC10 and TCR And TCRβ.

Whether before or after genetic modification of the T cell, the T cell is described, for example, in US Pat. No. 6,352,694; 6,534,055; 6,905. , 680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; The same No. 7,232,566; the same No. 7,175,843; the same No. 5,883,223; the same No. 6,905,874; the same No. 6,797, 514 can be activated and extended by generally using the methods as described in 6,867,041; and 7,572,631. T cells can expand in vitro or in vivo.

Unless otherwise indicated, the practice of the present invention utilizes prior arts of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA within the scope of the art in the art. .. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989) (Sambrook, Fritsch and Maniatis); MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012) (Green and Sambrook); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1987) (F.M Ausube, et al. Eds.); The series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (1995) (M.J. MacPherson, B.D. eds.); ANTIBODIES, A LABORATORY MANUAL (1988) (Harrow and Lane, eds.); ANTIBODIES A LABORATORY MANUAL, 2nd edition (2013) (EA Greenline) . I. Freshney, ed.).

In carrying out the present invention, unless otherwise specified, prior art for producing genetically modified mice is utilized. Martin H. See Hofker and Jan van Deursen, TRANSGENIC MOUSE METHODS AND PROTOCOLS, 2nd edition (2011).

ALS
US Patent Application Publication No. 20110023144 describes genetic alterations of cells, animals and proteins associated with amyotrophic lateral sclerosis (ALS) disease using zinc finger nucleases. ALS is characterized by the gradual and definite degeneration of certain nerve cells in the cortex, brainstem, and spinal cord involved in voluntary movements.

Motor neuron disorders and the proteins associated with them are a diverse collection of proteins that result in susceptibility to the development of motor neuron disorders, the presence of motor neuron disorders, the severity of motor neuron disorders, or any combination thereof. The present disclosure includes editing any chromosomal sequence that encodes a protein associated with ALS disease, a particular motor neuron disorder. Proteins associated with ALS are typically selected based on the experimental association of ALS-related proteins with ALS. For example, a population with ALS may have an increased or decreased production rate or circulating concentration of ALS-related proteins as compared to a population without ALS. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with ALS may be, but not limited to, genomic techniques including, but not limited to, DNA microarray analysis, gene expression linkage analysis (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). It can be identified by obtaining the gene expression profile of the encoding gene.

Non-limiting examples include, but are not limited to, ALS-related proteins include: SOD1 superoxide dismutase 1, ALS3 amyotrophic lateral sclerosis 3 SETX senataxin ALS5 amyotrophic lateral sclerosis: Sclerosis 5 FUS sarcoma fusion ALS7 amyotrophic lateral sclerosis 7 ALS2 amyotrophic lateral sclerosis DPP6 dipeptidyl peptidase 6 sclerosis 2 NEFH neurofilament, heavy PTGS1 prostaglandin-polypeptide endoperoxide synthase 1 SLC1A2 1 TNFRSF10B Tumor necrosis factor (glia high affinity receptor superfamily, glutamic acid transporter), member 10b member 2 PRPH periferin HSP90AA1 heat shock protein 90 kDa α (cytoplasmic type), class A member 1 GRIA2 glutamic acid receptor, IFNG interferon, γ Ion channel type, AMPA 2 S100B S100 calcium-binding FGF2 fibroblast growth factor 2 protein B AOX1 aldehyde oxidase 1 CS citrate synthase TARDBP TAR DNA-binding protein TXN thioredoxin RAPH1 Ras-related MAP3K5 Mitegen-activated protein (RaIGDS / AF) Kinase 5 Plextrin homologous domain 1 NBEAL1 Neurobactin-like 1 GPX1 Glutathion peroxidase 1 ICA1L Pancreatic islet cell self-antigen RAC1 ras-related C3 botulinum 1.69 kDa-like toxin substrate 1 MAPT microtube-related ITPR2 inositol 1,4,5-protein τ Acid receptor, type 2 ALS2CR4 amyotrophic lateral sclerosis GLS glutaminase sclerosis 2 (juvenile) chromosome region, candidate 4 ALS2CR8 amyotrophic lateral sclerosis 2 (juvenile) receptor chromosome region , Candidate 8 ALS2CR11 Amyotrophic lateral sclerosis FOLH1 Folic acid hydrolase 1 Sclerosis 2 (juvenile) chromosomal region, Candidate 11 FAM117B sequence Family P4HB prolyl 4-hydroxylase, similarity 117, member B β-polypeptide CNTF hairy neurotrophic factor SQSTM1 sequestosome 1 STRADB STE20-related kinase NAIP NLR family, apoptosis adapter β-inhibiting protein YWHAQ tyrosine 3-SLC33A1 solute Transport Carrier Family 33 Monooxygenase / Tryptoph (Acetyl-CoA Transporter), An 5-monooxygenase Member 1 Activated Protein, θ Polypeptide TRAK2 Transport Protein, FIG. 4 FIG. 4 Homolog, SAC1 Kinesin binding 2 Lipid phosphatase domain-containing NIF3L1 NIF3 NGG1 interaction INA Internexin neuron factor 3-like 1 Intermediate-diameter filament protein, α PARD3B par-3 partition COX8A Cytochrome c oxidase deficient 3 Homolog B subunit VIIIA CDK15 Kinases HECW1 HECT, C2 and WW 15 Domain-containing E3 ubiquitin protein ligase 1 NOS1 nitrogen monoxide synthase 1 MET met proto-oncogene SOD2 superoxide dismutase 2, HSBP1 heat shock 27 kDa mitochondrial protein 1 NEFL neurofilament, light CTSB cateptin B ANG angiogenin, HSPA8 heat shock 70 kDa ribonuclease, RNase A protein 8 family, 5 VAPB VAMP (follicle-ESR1 estrogen receptor 1 related membrane protein) related proteins B and C SNCA synuclein, α HGF hepatocellular growth factor CAT catalase ACTB actin , Β NEFM Neurofilament, Medium TH Tyrosine Hydroxylase Polypeptide BCL2 B Cell CLL / Lymphoma 2 FAS Fas (TNF Receptor Superfamily, Member 6) CASP3 Caspase 3, Apopulation-CLU Clusterlin-Related Cytine Peptidase SMN1 Motor Neurosurvival G6PD Glucose -6-phosphate 1, telomea dehydrogenase BAX BCL2-related X HSF1 heat shock transcription protein factor 1 RNF19A ring finger protein 19A JUN jun cancer gene ALS2CR12 muscle atrophic lateral cord HSPA5 heat shock 70 kDa sclerosis 2 (juvenile) protein 5 chromosome region , Candidate 12 MAPK14 Mightgen activating protein IL10 Interleukin 10 Kinases 14 APEX1 APEXnuclease TXNRD1 Thioredoxin reductase 1 (multifunctional DNA repair) Reenzyme) 1 NOS2 Nitrogen monoxide synthase 2, TIMP1 TIMP metallopeptidase inducible inhibitor 1 CASP9 caspase 9, apoptosis-X-chain inhibitor of XIAP related cysteine apoptosis peptidase GLG1 Gordiglycoprotein 1 EPO erythropoetin VEGN Factor A GDNF Glia cell-derived NFE2L2 Nuclear factor (erythrocyte-neurotrophic factor-derived 2) -like 2 SLC6A3 Solute transport carrier family 6 HSPA4 Heat shock 70 kDa (neurotransmitter protein 4 transporter, dopamine), member 3 APOE apolypoprotein E PSMB8 Proteasome (prosome, macropine) subunit, β-type, 8 DCTN1 dynactin 1 TIMP3 TIMP metallopeptidase inhibitor 3 KIFAP3 kinesin-related SLC1A1 solute transport carrier family 1 protein 3 (neuron / epithelial high-affinity glutamate transporter, system Xag), Member 1 SMN2 Motor Neuron Survival CCNC Cyclone C 2, Centromea MPP4 Membrane Protein, STUB1 STIP1 Homologous and U-Palmitylated 4 Box-Containing Protein 1 ALS2 Amyloid β (A4) PRDX6 Peroxyledoxin 6 Progenitor Protein SYP Synaptophysin CA 1 CASP1 caspase 1, apoptosis-GART phosphoribosylglycineami-related cysteine doformyltransferase, peptidase phosphoribosylglycineamido synthetase, phosphoribosylaminoimidazole synthetase CDK5 cyclin-dependent kinase 5 ATXN3 ataxin 3 RTN4 reticulone 4 C1 q Subcomponent, B chain VEGFC Nerve growth factor HTT Huntingtin receptor PARK7 Parkinson's disease 7 XDH xanthine dehydrogenase GFAP Glia fibrous acidic MAP2 microtubule binding protein tan Pact 2 CYCS cytochrome c, somatic cell type, FCGR3B IgG Fc fragment, low affinity IIIb, CCS copper chaperon UBL5 ubiquitin-like 5 superoxide dismutase MMP9 matrix metallopeptidase SLC18A3 solute transport carrier family 189 ((vesicular acetylcholine)) , Member 3 TRPM7 Transient receptor HSPB2 Heat shock 27 kDa Potential cation channel, Protein 2 Subfamily M, Member 7 AKT1 v-akt Mouse thymus adenoma DERL1 Der1-like domain family, Viral cancer gene Homolog 1 Member 1 CCL2 Chemokine (C- C motif) NGRN noigulin, neuroprosthetic ligand 2 Elongation-related GSR glutathione reductase TPPP3 Tubulin polymerization promoting protein family member 3 APAF1 Apoptotic peptidase BTBD10 BTB (POZ) domain activator 1 containing 10 GLUD1 glutamate CXCR4 chemo ) Dehydrogenase 1 Receptor 4 SLC1A3 Solute Transport Carrier Family 1 FLT1 fms Related Tyrosine (Glia High Affinity Glutamic Acid Transporter), Member 3 Kinase 1 PON1 Paraoxonase 1 AR Androgen Receptor LIF Leukemia Inhibitor ERBB3 v-erb-b2 Red Blast leukemia virus cancer gene homologue 3 LGALS1 lectin, galactoside-CD44 CD44 molecule binding, soluble, 1 TP53 tumor protein p53 TLR3 Toll-like receptor 3 GRIA1 glutamate receptor, GAPDH glyceraldehyde-3-ion channel type, AMPA 1 Dehydrogenase phosphate GRIK1 glutamate receptor, DES desmin ion channel type, chinic acid 1 CHAT choline acetyl transferase FLT4 fms-related tyrosine kinase 4 CHMP2B chromatin modified BAG1 BCL2-related protein 2B atano gene MT3 metallotionein 3 CHRNA α4 GSS glutathione synthetase BAK1 BCL2-antagonist / killer 1 KDR kinase insertion domain GSTP1 glutathione S-transferase receptor (type III π1 receptor tyrosine kinase) OGG1 8-oxoguanine DNA IL6 interleukin 6 (interferon, glycosylase β2).

Animals or cells have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disrupted chromosomal sequences encoding ALS-related proteins, and disrupted ALS-related proteins. Can include sequences integrated into 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomes encoding. Preferred ALS-related proteins include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (sarcoma fusion), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (Vascular Endothelial Growth Factor B), and VAGFC (Vascular Endothelial Growth Factor C), and any combination thereof are included.

Autism US Patent Application Publication No. 20110023145 describes genetic alterations of cells, animals and proteins associated with autism spectrum disorders (ASD) using zinc finger nucleases. Autism Spectrum Disorders (ASD) is a group of disorders characterized by qualitative disorders of social interaction and communication, as well as limited repetitive and stereotyped behaviors, interests, and activities. The three disorders, autism, Asperger's syndrome (AS) and pervasive developmental disorder (PDD-NOS), are a series of the same disorders with varying severity, associated intellectual function and medical condition. .. ASD is a predominantly genetically determined disorder with a heritability of about 90%.

U.S. Patent Application Publication No. 20110023145 includes editing any chromosomal sequence encoding a protein associated with ASD, which is applicable to the CRISPR Cas system of the invention. ASD-related proteins are typically selected based on the experimental association between ASD-related proteins and the incidence or symptoms of ASD. For example, a population with ASD may have an increased or decreased production rate or circulating concentration of ASD-related proteins compared to a population without ASD. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with ASD can be, but not limited to, those proteins using genomic techniques including DNA microarray analysis, gene expression linkage analysis (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). It can be identified by obtaining the gene expression profile of the encoding gene.

Non-limiting examples of medical conditions or disorders that may be associated with proteins associated with ASD include autism, Asperger's syndrome (AS), pervasive developmental disorder (PDD-NOS), Rett syndrome, tuberous sclerosis. Includes illness, phenylketonuria, Smith-Remli-Opitz syndrome and Fragile X syndrome. As a non-limiting example, proteins associated with ASD include, but are not limited to, the following proteins: ATP10C aminophospholipid-MET MET receptor transport ATPase tyrosine kinase (ATP10C) BZRAP1 MGLUR5 (GRM5) Metabolic glutamate Receptor 5 (MGLUR5) CDH10 Cadherin 10 MGLUR6 (GRM6) Metabolic glutamate receptor 6 (MGLUR6) CDH9 Cadherin 9 NLGN1 Neurolysine 1 CNTN4 Contactin 4 NLGN2 Neurolysine 2 CNTNAP2 Contactin-like 7-Dehydrocholesterol NLGN4X Neurolysine 4 X-Reductase (DHCR7) Linked DOC2A Double C2-like Domain-NLGN4Y Neurolysine 4 Y-Containing Protein α Linked DPP6 Dipeptidyl NLGN5 Neurolysine 5 Aminopeptidase 2 ) NRCAM Nerve Cell Adhesion Molecular (NRCAM) MDGA2 Vulnerable X Mental Delay NRXN1 Nurexin 1 1 (MDGA2) FMR2 (AFF2) AF4 / FMR2 Family Member 2 OR4M2 Smell Receptor (AFF2) 4M2 FOXP2 Forkhead Box Protein P2 OR4 (FOXP2) 4N4 FXR1 Vulnerable X Mental OXTR Oxytocin Receptor Delay, Autosomal (OXTR) Homolog 1 (FXR1) FXR2 Vulnerable X Mental PAH Phenylalanine Delay, Autosomal Hydroxidease (PAH) Homolog 2 (FXR2) GABRA1 Aminobutyric acid PTEN phosphatase and receptor subunit α-1 Tensin homolog (GABRA1) (PTEN) GABRA5 GABAA (γ-aminobuty PTPRZ1 receptor-type acid) Receptor α5 Tyrosine protein subunit (GABRA5) Phosphatase ζ (PTPRZ1) GABRB1 γ- Aminobutyric acid LERN receptor -Lin receptor subunit β-1 (GABRB1) GABRB3 GABAA (γ-aminodairy RPL10 60S ribosome acid) receptor β3 subunit Protein L10 (GABRB3) GABRG1 γ-aminobutyric acid SEMA5A semaphorin-5A receptor subunit γ-1 (SEMA5A) (GABRG1) HIRI P3 HIRA interacting protein 3 SEZ6L2 attack-related 6 homolog (mouse) -like 2 HOXXA1 homeobox protein Hox-A1 SHANK3 SH3 and multiple (HOXXA1) ankyrin repeat domains 3 (SHANK3) RHAK3 And multiple anquilin repeat domains 3 (SHBZRAP1) LAMB1 laminin subunit β-1 SLC6A4 serotonin (LAMB1) transporter (SERT) MAPK3 mitogen activating protein TAS2R1 taste receptor kinase 3 type 2 member 1 TAS2R1 MAZ-related Sexual sclerosis protein protein 1 MDGA2 MAM domain-containing TSC2 nodular sclerosis glycosylphosphatidylinositol protein 2 anchor 2 (MDGA2) MECP2 methyl CpG binding UBE3A ubiquitin protein protein 2 (MECP2) ligase E3A (UBE3A) ligase E3A (UBE3A) Protein 2 (MECP2) MMTV integration site family, member 2 (WNT2).

The identities of the ASD-related proteins whose chromosomal sequences are edited may and will be different. In a preferred embodiment, the ASD-related protein whose chromosomal sequence is edited is the benzodiazepine receptor (peripheral) -related protein 1 (BZRAP1) encoded by the BZRAP1 gene, the AFF2 gene (also referred to as MFR2). AF4 / FMR2 family member 2 protein encoded by (AFF2), fragile X mental retardation autosomal homologue 1 protein encoded by the FXR1 gene (FXR1), fragile X mental retardation autosomal homologue 2 encoded by the FXR2 gene Protein (FXR2), MAM domain-containing glycosylphosphatidylinositol anchor 2 protein encoded by MDGA2 gene (MDGA2), methyl CpG binding protein 2 encoded by MECP2 gene (MECP2), MGLUR5-1 gene (also referred to as GRM5) It can be the metabolite glutamate receptor 5 (MGLUR5) encoded by, the neurexin 1 protein encoded by the NRXN1 gene, or the semaphorin 5A protein (SEMA5A) encoded by the SEMA5A gene. In an exemplary embodiment, the animal undergoing genetic modification is a rat, and the edited chromosomal sequences encoding proteins associated with ASD are listed below: BZRAP1 benzodiazapine receptor XM_0027277789, (peripheral) related XM_213427. , Protein 1 (BZRAP1) XM_002724533, XM_001081125 AFF2 (FMR2) AF4 / FMR2 Family Member 2 XM_219832, (AFF2) XM_001054673 FXR1 Fragile X Mental NM_001012179 Delayed, Autosomal Homolog 1 (FXR1) Homolog 2 (FXR2) MDGA2MAM Domain Containing NM_199269 Glycophosphatidylinositol Anchor 2 (MDGA2) MECP2 Methyl CpG Bonded NM_022673 Protein 2 (MECP2) MGLUR5 Metabolic Glutamic Acid NM_017012 (GRM5) NM_01673 5A (SEMA5A) NM_0011076559.

Trinucleotide Repeat Elongation Disorder (TRE)
US Patent Application Publication No. 2011016540 describes genetic modification of cells, animals and proteins associated with trinucleotide repeat elongation disorders using zinc finger nucleases. Trinucleotide repeat elongation disorders are complex progressive disorders involving developmental neurobiology and often affecting cognitive and sensorimotor function.

Trinucleotide repeat elongation proteins are a diverse set of proteins associated with susceptibility to the development of trinucleotide repeat elongation disorders, the presence of trinucleotide repeat elongation disorders, the severity of trinucleotide repeat elongation disorders, or any combination thereof. .. Trinucleotide repeat elongation disorders are divided into two types, which are determined by the type of repeat. The most common repeat is the triplet CAG, which encodes the amino acid glutamine (Q) when present in the coding region of a gene. Therefore, these disorders are referred to as polyglutamine (polyQ) disorders and include the following disorders: Huntington's disease (HD); spinal and bulbar muscular atrophy (SBMA); spinocerebellar ataxia (SCA1, type 2, type 3, 3) Type, Type 6, Type 7, and Type 17); and Dentatorubral-red nucleus paleosphere Louis body ataxia (DRPLA). The remaining trinucleotide repeat elongation disorders are referred to as non-polyglutamine disorders because the CAG triplet is not involved or the CAG triplet is not in the coding region of the gene. Non-polyglutamine disorders include fragile X syndrome (FRAXA); fragile XE mental retardation (FRAXE); Friedreich's ataxia (FRDA); myotonic dystrophy (DM); and spinocerebellar ataxia (SCA8 and 12). ) Is included.

Proteins associated with trinucleotide repeat elongation disorders are typically selected based on the experimental association between proteins associated with trinucleotide repeat elongation disorders and trinucleotide repeat elongation disorders. For example, a population with a trinucleotide repeat elongation disorder may have an increased or decreased production rate or circulating concentration of a protein associated with a trinucleotide repeat elongation disorder as compared to a population without a trinucleotide repeat elongation disorder. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with trinucleotide repeat elongation disorders can be, but not limited to, using genomic techniques including, but not limited to, DNA microarray analysis, gene expression linkage analysis (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). It can be identified by obtaining the gene expression profile of the genes encoding those proteins.

Non-limiting examples of proteins associated with trinucleotide repeat elongation disorders include AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (huntingtin), DMPK (muscle tonic dystrophy protein kinase), FXN (Frataxin), ATXN2 (Ataxin 2), ATN1 (Atrophin 1), FEN1 (Flap structure specific endonuclease 1), TNRC6A (Trinucleotide repeat containing 6A), PABPN1 (Poly (A) binding protein, Nucleus 1), JPH3 (Junktophyllin 3), MED15 (Mediator Complex Subunit 15), ATXN1 (Ataxin 1), ATXN3 (Ataxin 3), TBP (TATA Box Binding Protein), CACNA1A (Calcium Channel, Potential Dependent, P / Q Type, α1A subunit), ATXN80S (ATXN8 reverse chain (non-protein code)), PPP2R2B (protein phosphatase 2, regulatory subunit B, β), ATXN7 (ataxin 7), TNRC6B (trinucleotide repeat containing 6B), TNRC6C (6C containing trinucleotide repeat), CELF3 (CUGBP, Elav-like family member 3), MAB21L1 (mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2, colon cancer, non-polyposis 1) Type (E. coli)), TMEM185A (transmembrane protein 185A), SIX5 (SIX homeobox 5), CNPY3 (canopy 3 homolog (zebrafish)), FRAX (fragile site, folic acid type, rare, fla ( X) (q28) E), GNB2 (guanine nucleotide binding protein (G protein), β polypeptide 2), RPL14 (ribosome protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR (transsiletin) ), EP400 (E1A binding protein p400), GIGYF2 (GRB10 interacting GYF protein 2), OGG1 (8-oxoguanine DNA glycosylase), STC1 (staniocalcin 1), CNDP1 (carnosindipeptidase 1 (metallopeptidase M20 family)), C10orf2 (protein 10 open reading frame 2), MAML3 mastermind-like 3 (Drosophila), DK C1 (congenital keratoderma 1, dyskelin), PAXIP1 (PAX (with transcriptional activation domain) interacting protein 1), CASK (calcium / calmodulin-dependent serine protein kinase (MAGUK family)), MAPT (microtubes) Binding protein τ), SP1 (Sp1 transcription factor), POLG (polymerizer (DNA-oriented), γ), AFF2 (AF4 / FMR2 family, member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein 1), ABT1 (basal transcription activator 1), KLK3 (calicrane-related peptidase 3), PRNP (prion protein), JUN (jun cancer gene), KCNN3 (Potasium intermediate / small conductance calcium activation channel, subfamily N, member 3), BAX (BCL2-related X protein), FRAXA (fragile site, folic acid type, rare, fla (X) (q27.3) A (giant) (Pyoderma, mental retardation)), KBTBD10 (10 containing Kerhi repeat and BTB (POZ) domains), MBNL1 (Muscle blind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E. coli (E. coli)) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1 (elongated repeat domain, CAG / CTG 1), TSC1 (nodular sclerosis 1), COMP ( Cartilage oligomer matrix protein), GCLC (glutamic acid-cysteine ligase, catalytic subunit), RRAD (Ras associated with diabetes), MSH3 (mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2) , CD44 (CD44 molecule (Indian blood type)), CTCF (CCCTC binding factor (zinc finger protein)), CCND1 (cycline D1), CLSPN (claspin homolog (Xenopus laevis)), MEF2A (muscle cell) Enhancer factor 2A), PTPRU (protein tyrosine phosphatase, receptor type, U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (with tripartite motif 22), WT1 (Wilms tumor 1), AHR (aryl) Hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine S-methyltransferase), NDP (Norie's disease (pseudoglioma)), ARX (Aristales-related homeobox), MUS81 (MUS81 endonuclease homolog (S) S. cerevisiae)), TYR (tyrosinase (eye cutaneous leukoderma IA)), EGR1 (early growth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homolog (Drosophila)) Like), FABP2 (fatty acid binding protein 2, intestine), EN2 (engraved homeobox 2), CRYGC (crystallin, γC), SRP14 (signal recognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB (crystalin, γB) , PDCD1 (programmed cell death 1), HOXXA1 (homeobox A1), ATXN2L (ataxin 2-like), PMS2 (PMS2 post-mitotic segregation increased type 2 (S. cerevisiae)), GLA (galactosidase, α) ), CBL (Cas-Br-M (mouse) ecotropic retrovirus transformation sequence), FTH1 (feritin, heavy polypeptide 1), IL12RB2 (inter) -Leukin 12 receptor, β2), OTX2 (orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerizer (DNA directional), γ2, accessory subunit), DLX2 (distalless homeobox 2), SIRPA (Signal Regulatory Protein α), OTX1 (Orthodenticle Homeobox 1), AHRR (aryl hydrocarbon receptor repressor), MANF (Middle brain stellate cell-derived neurotrophic factor), TMEM158 (Transmembrane protein 158 (gene) / Pseudogene)), and ENSG0000000768687.

Preferred proteins associated with trinucleotide repeat elongation disorders include HTT (huntingtin), AR (androgen receptor), FXN (frataxin), Atxn3 (ataxin), Atxn1 (ataxin), Atxn2 (ataxin), Atxn7 ( Ataxin), Atxn10 (Ataxin), DMPK (Muscle Tonic Dystrophy Protein Kinase), Atn1 (Atrophin 1), CBP (CREB Binding Protein), VLDLR (Very Low Density Lipoprotein Receptor), and any combination thereof. Be done.

Alzheimer's Disease US Patent Application Publication No. 20110023153 describes genetic modification of cells, animals and proteins associated with Alzheimer's disease using zinc finger nucleases. Once modified cells and animals are targeted mutations using the scales commonly used in AD testing-eg, without limitation, learning and memory, anxiety, depression, addiction, and sensorimotor function. Can be further tested using known methods for studying the effects of s on onset and / or progression of AD, as well as assays that measure behavioral, functional, pathological, metabolic and biochemical functions.

The present disclosure includes editing any chromosomal sequence that encodes a protein associated with AD. AD-related proteins are typically selected based on the experimental association between AD-related proteins and AD disorders. For example, in a population with AD disorder, the production rate or circulating concentration of AD-related proteins may increase or decrease as compared to the population without AD disorder. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, AD-related proteins encode those proteins using genomic techniques including, but not limited to, DNA microarray analysis, gene expression linkage analysis (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). It can be identified by obtaining the gene expression profile of the gene.

Examples of Alzheimer's disease-related proteins include, for example, the ultra-low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, the ubiquitin-like modifier activator 1 (UBA1) encoded by the UBA1 gene, or the UBA3 gene. NEDD8 activating enzyme E1 catalytic subunit protein (UBE1C) encoded by.

As a non-limiting example, proteins associated with AD include, but are not limited to, proteins listed below: the protein encoded by the chromosomal sequence ALAS2 Δ-aminolevulinate synthase 2 (ALAS2) ABCA1 ATP. Binding Cassette Transporter (ABCA1) ACE Angiotensin I-Converter (ACE) APOE Apolipoprotein E Progenitor (APOE) APP Amyloid Progenitor Protein (APP) AQP1 Aquaporin 1 Protein (AQP1) BIN1 Myc Box Dependent Interaction Protein 1 or Cross-linking Integrator 1 protein (BIN1) BDNF Brain-derived neurotrophic factor (BDNF) BTNL8 Butylovylin-like protein 8 (BTNL8) C1ORF49 Chromium 1 Open reading frame 49 CDH4 Cadherin 4 CHRNB2 neuron acetylcholine 4 CHRNB2 MARVEL transmembrane domain-containing protein 2 (CKLFSF2) CLEC4E C-type lectin domain family 4, member e (CLEC4E) CLU cluster phosphorus protein (also known as apoplipoprotein J) CR1 erythrocyte complement receptor 1 (CR1, also (Also known as CD35, C3b / C4b and immunoadhesive receptors) CR1L Erythrocyte complement receptor 1 (CR1L) CSF3R Granulocyte colony stimulating factor 3 receptor (CSF3R) CST3 Cystatin C or Cystatin 3 CYP2C Cytochrome P450 2C DADK1 Cell Death-Related Protein Kinase 1 (DACK1) ESR1 Estrogen Receptor 1 FCAR IgA Receptor Fc Fragment (FCAR, also known as CD89) FCGR3B IgG Fc Fragment, Low Affinity IIIb, Receptor (FCGR3B or CD16b) FFA2 Free fatty acid receptor 2 (FFA2) FGA fibrinogen (factor I) GAB2 GRB2-related binding protein 2 (GAB2) GAB2 GRB2-related binding protein 2 (GAB2) GALP galanin-like peptide GAPDHS Glyceraldehyde-3-phosphate dehydrogenase, sperm formation (GAPDHS) GMBP GMBP HP haptoglobin (HP) HTR7 5-hydroxytryptamine (serotonin) receptor 7 (adenylate cyclase conjugated type) IDE insulin degrading enzyme IF127 IF127 IFI6 interferon, α -Inducible protein 6 (IFI6) IFIT2 Interferon-inducing protein 2 with tetratricopeptide repeat (IFIT2) IL1RN Interleukin-1 receptor antagonist (IL-1RA) IL8RA Interleukin 8 receptor, α (IL8RA or CD181) IL8RB Interleukin 8 receptor, β (IL8RB) JAG1 Jagd 1 (JAG1) KCNJ15 potassium inward rectifying channel, subfamily J, member 15 (KCNJ15) LRP6 low density lipoprotein receptor related protein 6 (LRP6) MAPT microtubule binding Protein τ (MAPT) MARK4 MAP / Microtube Affinity Regulatory Kinase 4 (MARK4) MPHOSPH1 M Phase Phosphorin Protein 1 MTHFR 5,10-Methylenetetrahydrofolate Reductor MX2 Interferon-Induced GTP-Binding Protein MX2 NBN Nibrin, also known as NCSTN Nicastrin NIACR2 Niacin receptor 2 (NIACR2, also known as GPR109B) NMNAT3 Nicotinamide nucleotide Adenyl transferase 3 NTM Neurotrimin (or HNT) ORM1 Orosmucoid 1 (ORM1) or α-1-acid protein 1 P2RY13 P2Y Purine Receptor 13 (P2RY13) PBEF1 Pre-B cell colony enhancer 1 (PBEF1) or nicotinamide phosphoribosyl transferase (NAmPRTase or Nampt) also known as bisfatin PCK1 phosphoenolpyruvate carboxykinase PICALM phosphatidylinos Aggregate protein (PICALM) PLAU urokinase type plasminogen activator (PLAU) PLXNC1 Plexin C 1 (PLXNC1) PRNP prion protein PSEN1 presenirin 1 protein (PSEN1) PSEN2 presenirin 2 protein (PSEN2) PTPRA protein tyrosine phosphatase receptor type A protein (PTPRA) RALGPS2 PH domain and RALGEF2 (RALGPS2) with SH3 binding motif Signal transduction-like regulator 2 (RGSL2) SELENBP1 selenium-binding protein 1 (SELNBP1) SLC25A37 Mitoferin 1 SORL1 Soltilin-related receptor L (DLR class) A repeat-containing protein (SORL1) TF transferase TFAM mitochondrial transcription factor A TNF tumor Tumor necrosis factor receptor superfamily member 10C (TNFRSF10C) TNFSF10 Tumor necrosis factor receptor superfamily, (TRAIL) member 10a (TNFSF10) UBA1 Ubiquitin-like modifier activator 1 (UBA1) UBA3 NEDD8 activator E1 catalytic subunit Protein (UBE1C) UBB Ubikitin B Protein (UBB) UBQLN1 Ubikirin 1 UCHL1 Ubikitin carboxyl-terminal esterase L1 protein (UCHL1) UCHL3 Ubikitin carboxyl-terminal hydrolase isozyme L3 protein (UCHL3) VLDL protein (UCHL3)

In an exemplary embodiment, the AD-related protein whose chromosomal sequence is edited is the ultra-low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, ubiquitin-like modifier activation encoded by the UBA1 gene. Enzyme 1 (UBA1), NEDD8 activator E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene, aquaporin 1 protein (AQP1) encoded by the AQP1 gene, ubiquitin carboxyl-terminated esterase L1 protein encoded by the UCHL1 gene. (UCHL1), ubiquitin carboxyl-terminated hydrolase isozyme L3 protein encoded by the UCHL3 gene (UCHL3), ubiquitin B protein encoded by the UBB gene (UBB), microtubule binding protein τ (MAPT) encoded by the MAPT gene, PTPRA Gene-encoded protein Tyrosine phosphatase receptor type A protein (PTPRA), phosphatidylinositol-binding clasulin assembly protein (PICALM) encoded by the PICALM gene, cluster phosphorus protein (apoplipoprotein) J encoded by the CLU gene (Also known as), Presenirin 1 protein encoded by the PSEN1 gene, Presenirin 2 protein encoded by the PREN2 gene, Soltilin-related receptor L (DLR class) A repeat-containing protein (SORL1) protein encoded by the SORL1 gene, It can be an amyloid precursor protein (APP) encoded by the APP gene, an apolipoprotein E precursor (APOE) encoded by the APOE gene, or a brain-derived neurotrophic factor (BDNF) encoded by the BDNF gene. In an exemplary embodiment, the animal undergoing genetic modification is a rat, and the edited chromosomal sequence encoding a protein associated with AD is: APP Amyloid Progenitor Protein (APP) NM_019288 AQP1 Aquaporin 1 Protein (AQP1) NM_012778 BDNF Brain-derived neurotrophic factor NM_012513 CLU cluster phosphorus protein (also known as NM_053021 apoplipoprotein J) MAPT microtubule binding protein NM_017212 τ (MAPT) PICALM phosylin PSEN1 Presenirin 1 protein (PSEN1) NM_019163 PSEN2 Presenirin 2 protein (PSEN2) NM_031087 PTPRA protein Tyrosine phosphatase NM_012763 Receptor type A protein (PTPRA) SORL1 Soltilin-related receptor L (DLR XM_217115 UBA1 Ubikitin-like modifier activation NM_001014080 Enzyme 1 (UBA1) UBA3 NEDD8 Activator E1 NM_057205 Catalytic subunit protein (UBE1C) UBB Ubikitin B protein (UBB) Ubikitin B protein (UBB) Ubikitin B protein (UBB) NM_13895 Terminal NM_001110165 Hydrolase isozyme L3 protein (UCHL3) VLDLR Ultra-low density lipoprotein NM_013155 Receptor protein (VLDLR).

The animal or cell was disrupted by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more encoding proteins associated with AD. Chromosome sequences and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more chromosomes encoding proteins associated with AD It may contain an integrated sequence.

The chromosomal sequence that is edited or integrated can be modified to encode a protein associated with altered AD. Numerous mutations in AD-related chromosomal sequences have been associated with AD. For example, a V7171 (ie, valine at position 717 is converted to isoleucine) missense mutation in APP causes familial AD. Multiple mutations in the presenilin 1 protein, such as H163R (ie, histidine at position 163 is converted to arginine), A246E (ie, alanine at position 246 is converted to glutamic acid), L286V (ie, leucine at position 286 is converted to valine) and C410Y. (Ie, cysteine at position 410 is converted to tyrosine) causes familial Alzheimer type 3. Mutations in the presenilin 2 protein, such as N141I (ie, asparagine at position 141 is converted to isoleucine), M239V (ie, methionine at position 239 is converted to valine), and D439A (ie, aspartic acid at position 439 is converted to alanine) are family members. Causes sex Alzheimer's type 4. Other associations between genetic variation of AD-related genes and disease are known in the art. For example, Waring et al. (2008) Arch. Neurol. 65: 329-334, which disclosure is incorporated herein by reference in its entirety.

Examples of disease-related genes Examples of disease-related genes and polynucleotides are listed in Tables A and B. Examples of biochemical signaling pathway-related genes and polynucleotides are listed in Table C.

List of exemplary target genes, target loci, and target polynucleotides As an example, the chromosome sequence is, but is not limited to, IL1B (interleukin 1, β), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS ( Prostaglandin 12 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoetin 1), ABCG8 (ATP binding cassette, subfamily G (WHITE), member 8), CTSK ( Catepsin K), PTGIR (prostacyclin 12 (prostacyclin) receptor (IP)), KCNJ11 (potassium inward rectifying channel, subfamily J, member 11), INS (insulin), CRP (C-reactive protein, Pentraxin-related), PDGFRB (platelet-derived growth factor receptor, β-polypeptide), CCNA2 (cyclin A2), PDGFB (platelet-derived growth factor β-polypeptide (salcarcinoma virus (v-sis) cancer gene homologue)), KCNJ5 ( Potassium inward rectifying channel, subfamily J, member 5), KCNN3 (potassium intermediate / small conductance calcium activation channel, subfamily N, member 3), CAPN10 (calpine 10), PTGES (prostacyclin E synthase) , ADRA2B (adrenalinergic, α-2B-, receptor), ABCG5 (ATP binding cassette, subfamily G (WHITE), member 5), PRDX2 (peroxyredoxin 2), CAPN5 (calpine 5), PARP14 (poly) (ADP-ribose) polymerase family, member 14), MEX3C (mex-3 homolog C (C. elegans)), ACE angiotensin I-converting enzyme (peptidyl dipeptidase A) 1), TNF (tumor necrosis factor) (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon, β2)), STN (statin), SERPINE1 (cellpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), Members 1), ALB (albumin), ADIPOQ (containing adiponectin, C1Q and collagen domain), APOB (apolipoprotein B (including Ag (x) antigen)), APOE (apolipotampa) Quality E), LEP (Leptin), MTHR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (Apolipoprotein AI), EDN1 (Endocerin 1), NPBP (Natriuretic peptide precursor B) , NOS3 (nitrogen monoxide synthase 3 (endothelial cells)), PPARG (peroxysome growth factor activated receptor γ), PLAT (plasminogen activator, tissue), PTGS2 (prostaglandin endoperoxide synthase 2 (prostaglan) Zin G / H synthase and cyclooxygenase)), CETP (cholesteryl ester transfer protein, plasma), AGTR1 (angiotensin II receptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-coase A reductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE (selectin E), REN (renin), PPARA (peroxysome growth factor activated receptor α), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2 (chemokine (CC motif) ligand 2), LPL (lipoprotein lipase), VWF (von Villebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (Transformation Growth Factor, β1), NPPA (Natriuretic Peptide Precursor A), IL10 (Interleukin 10), EPO (Erythropoetin), SOD1 (Super Oxide Dismutase 1, Soluble), VCAM1 (Vascular Cell Adhesion Molecule 1), IFNG (interferon, γ), LPA (lipoprotein, Lp (a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1), MAPK1 (mitogen-activated protein kinase 1), HP (haptoglobin), F3 (coagulation) Factor III (thromboplastin, tissue factor)), CST3 (cystatin C), COG2 (component 2 of oligomeric gorgi complex), MMP9 (matriuretic metallopeptidase 9 (zelatinase B, 92 kDa zeratinase, 92 kDa type IV collagenase)), SERPINC1 (Serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, coagulation promoting component), HMOX1 (hemoxygenase (decycling) 1), APOC3 (apolipoprotein C-III) , IL8 (interleukin 8), PROK1 (prokineticin 1), CBS (cystathionin β synthase), NOS2 (nitrogen monoxide synthase 2, inducible), TLR4 (Toll-like receptor 4), SELP (selectin P (granule)) Membrane protein 140 kDa, antigen CD62)), ABCA1 (ATP binding cassette, subfamily A (ABC1), member 1), AGT (angiotensinogen (selpin peptidase inhibitor, clade A, member 8)), LDLR (low density lipo) Protein receptor), GPT (Glutamic acid pyruvate transaminase (alanine aminotransferase)), VEGFA (vascular endothelial growth factor A), NR3C2 (nuclear receptor subfamily 3, group C, member 2), IL18 (interleukin 18 (interleukin 18) Interferon-γ inducer)), NOS1 (nitrogen monoxide synthase 1 (nerve type)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrinogen β chain) ), HGF (hepatocellular growth factor (hepapoetin A; scattering factor)), IL1A (interleukin 1, α), RETN (receptor), AKT1 (v-akt mouse thoracic adenoma virus cancer gene homologue 1), LIPC (lipase, Liver), HSPD1 (heat shock 60 kDa protein 1 (chaperonin)), MAPK14 (mitogen activated protein kinase 14), SPP1 (secretory phosphorus protein 1), ITGB3 (integrin, β3 (platelet glycoprotein 111a, antigen CD61)), CAT (Catalase), UTS2 (urotensin 2), THBD (thrombomodulin), F10 (coagulation factor X), CP (celluloplasmin (ferroxydase)), TNFRSF11B (tumor necrosis factor receptor superfamily, member 11b), EDNRA (endoserine) Receptor type A), EGFR (epithelial growth factor receptor (erythroblastic leukemia viral (v-erb-b) cancer gene homologue, bird)), MMP2 (matrix metallopeptidase 2 (zelatinase A, 72 kDa zeratinase, 72 kDa)) IV type collagenase)), PLG (plasminogen), NPY (nerve peptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis) Viral cancer gene homolo Gu (tri)), FN1 (fibronectin 1), CMA1 (chimase 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide binding protein (G protein), β-polypeptide 3), ADRB2 (Adrenalinergic, β-2-, receptor, surface), APOA5 (apolypoprotein AV), SOD2 (superoxide dismutase 2, mitochondria), F5 (coagulation factor V (proacceleline, unstable factor)) ), VDC (Vitamin D (1,25-dihydroxyvitamin D3) receptor), ALOX5 (arachidonate 5-lipoxygenase), HLA-DRB1 (major tissue compatible gene complex, class II, DR β1), PARP1 (poly) (ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2), AGER (terminal glycated product-specific receptor), IRS1 (insulin receptor substrate 1), PTGS1 (prostaglandin endoperoxide) Syntase 1 (prostaglandin G / H synthase and cyclooxygenase), ECE1 (endoserine converting enzyme 1), F7 (coagulation factor VII (serum prothrombin conversion promoter)), URN (interleukin 1 receptor antagonist), EPHX2 (Epoxide hydrolase 2, cytoplasm), IGFBP1 (insulin-like growth factor binding protein 1), MAPK10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily, member 6)), ABCB1 (ATP binding cassette, Subfamily B (MDR / TAP), member 1), JUN (jun cancer gene), IGFBP3 (insulin-like growth factor binding protein 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP specific), AGTR2 (angiotensin) II receptor, type 2), CD40 (CD40 molecule, TNF receptor superfamily member 5), LCAT (resitin cholesterol acyltransferase), CCR5 (chemokine (CC motif) receptor 5), MMP1 (matrix metallopeptidase 1) (Interstitial collagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM (adrenomedurin), DYT10 (dystony 10), STAT3 (signal transduction and transcriptional activator 3 (acute phase anti)) (Receptor)), MMP3 (matrix metallopeptidase 3 (stromelaicin 1, proseratinase)), ELN (elastin), USF1 (upstream transcription factor 1), CFH (complement factor H), HSPA4 (heat shock 70 kDa protein 4) ), MMP12 (matrix metallopeptidase 12 (macrophages elastase)), MME (membrane metalloendopeptide), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (catepsin B), ANXA5 (anexin) A5), ADRB1 (adrenalinergic, β-1-, receptor), CYBA (cytochrome b-245, α polypeptide), FGA (fibrinogen α chain), GGT1 (γ-glutamyltransferase 1), LIPG (lipase, Endophilic), HIF1A (hypoxic inducer 1, α-subunit (basic helix-loop-helix transcription factor)), CXCR4 (chemokine (C-X-C motif) receptor 4), PROC (protein C (coagulation order) Va factor and VIIIa factor inactivator)), SCARB1 (scavenger receptor class B, member 1), CD79A (CD79a molecule, immunoglobulin-related α), PLTP (phospholipid transfer protein), ADD1 (aducine 1 (α)) )), FGG (fibrinogen γ chain), SAA1 (serum amyloid A1), KCNH2 (potassium potential open channel, subfamily H (eg-related), member 2), DPP4 (dipeptidyl peptidase 4), G6PD (glucose-6) -Phosphate dehydrogenase), NPR1 (sodium diuretic peptide receptor A / guanylate cyclase A (atrial sodium diuretic peptide receptor A)), VTN (bitronectin), KIAA0101 (KIAA0101), FOS (FBJ mouse osteosarcoma virus cancer gene) Homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolyl isomerase G (cyclophyllin G)), IL1R1 (interleukin 1 receptor, type I), AR (androgen receptor), CYP1A1 (cytochrome P450, family) 1, Subfamily A, Polypeptide 1), SERPINA1 (Serpin peptidase inhibitor, Clade A (α-1 antiproteinase, antitrypsin), Member 1), MTR (5-Methyltetrahydrofolate homocysteine methyltransferase) Z), RBP4 (retinol binding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (cyclin-dependent kinase inhibitor 2A (inhibits melanoma, p16, CDK4)), FGF2 (fibroblast growth factor 2) (Basic)), EDNRB (endothelin receptor type B), ITGA2 (integrin, α2 ()
CD49B, VLA-2 receptor α2 subunit)), CABIN1 (calcinulin binding protein 1), SHBG (sex hormone binding globulin), HMGB1 (high mobility group box 1), HSP90B2P (heat shock protein 90 kDa β (Grp94)) , Member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4), GJA1 (gap binding protein, α1, 43 kDa), CAV1 (caveolin 1, caveora protein, 22 kDa), ESR2 ( Estrogen receptor 2 (ER β)), LTA (phosphotoxin α (TNF superfamily, member 1)), GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, sub Family D, Polypeptide 6), NGF (Nerve Growth Factor (β Polypeptide)), SP1 (Sp1 Transcription Factor), TGIF1 (TGFB Inducible Factor Homeobox 1), SRC (v-src sarcoma (Schmidt-Lupin A-) 2 (Schmidt-Ruppin A-2) virus cancer gene homologue (tri)), EGF (epithelial growth factor (β-urogastron)), PIK3CG (phosphoinositide-3-kinase, catalyst, γ-polypeptide), HLA-A ( Major histocompatibility gene complex, class I, A), KCNQ1 (potassium potential open channel, KQT-like subfamily, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (choline) Kinase α), BEST1 (bestrophin 1), APP (amyloid β (A4) precursor protein), CTNNB1 (catenin (cadherin-related protein), β1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombo)) Spondin receptor)), PRKAB1 (protein kinase, AMP activation, β1 non-catalyzed subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7 family, member A1), CX3CR1 (chemocaine (C-X3-C motif)) ) Receptor 1), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferase), HFE (hemochromatosis), IL17A (interleukin 17A), PTEN (phosphatase)・Tensin homolog), GSTM1 (glutathione S-transferase μ1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1 (coagulation factor XIII, A1 polypeptide), TTR (transsiletin), FABP4 (fatty acid binding protein) 4. Fat cells), PON3 (paraoxonase 3), APOC1 (apolipoprotein CI), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine) (Serotonin) receptor 2A), CSF3 (colony stimulator 3 (granulocytes)), CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11, Subfamily B, Polypeptide 2), PTH (parathyroid hormone), CSF2 (colony stimulator 2 (granulocyte macrophages)), KDR (kinase insertion domain receptor (type III receptor tyrosine kinase)), PLA2G2A (phospholipase A2) , Group IIA (platelet, lubricant)), B2M (β-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2) Family (mitomiloid)), TCF7L2 (transcription factor 7-like 2 (T cell-specific, HMG box)), BDKRB2 (brazikinin receptor B2), NFE2L2 (nuclear factor (erythrocyte-derived 2) -like 2), NOTCH1 (Notch homolog) 1. Translocation-related (Drosophila)), UGT1A1 (UDP glucuronosyl transferase 1 family, polypeptide A1), IFNA1 (interferon, α1), PPARD (peroxysome growth factor activating receptor δ), SIRT1 ( Sirtuin (silent mating type information regulation 2 homolog) 1 (S. S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1 (yellow body-forming-releasing hormone)), PAPPA (pregnancy-related plasma protein A, papalicin 1), ARR3 (alestin 3, retinal (X-alestin)), NPPC ( Sodium diuretic peptide precursor C), AHSP (α-hemoglobin stabilizing protein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13), MTOR (mechanical target of rapamycin (serine / threonine kinase)), ITGB2 ( Integrin, β2 (complementary component 3 receptors 3 and 4 subunits), GSTT1 (glutathione S-transferase θ1), IL6ST (interleukin 6 signaling factor (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase) B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocellular nuclear factor 4, α), SLC6A4 (solute transport carrier family 6 (neurotransmitter transporter, cellotonin)) , Member 4), PLA2G6 (phospholipase A2, VI group (cytoplasmic type, calcium independent)), TNFSF11 (tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solute transport carrier family 8 (sodium / calcium exchange)) Body), member 1), F2RL1 (coagulation factor II (thrombin) receptor-like 1), AKR1A1 (ald-ketreductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase 9 family, member A1), BGLAP (bone γ-carboxyglutamic acid (gla) -containing protein), MTTP (microsome triglyceride transfer protein), MTRR (5-methyltetrahydrofolate homocysteine methyltransferase reductase), SULT1A3 (sulfotransferase family, cytoplasmic type, 1A, phenol selection, Members 3), RAGE (renal tumor antigen), C4B (complementary component 4B (Chido blood type), P2RY12 (purine receptor P2Y, G protein conjugate, 12), RNLS (linalase, FAD-dependent amine oxidase) , CREB1 (cAMP response element binding protein 1), POMC (proopiomelanocortin), RAC1 (ras Related C3 botulinum toxin substrate 1 (rho family, low molecular weight GTP binding protein Rac1)), LMNA (Lamine NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, potential opening type, V type, α sub) Unit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), MIF (macrophage migration inhibitor (glycosylation inhibitor)), MMP13 (matrix metallopeptidase 13 (collagenase 3)), TIMP2 (TIMP metallo) Peptidase inhibitor 2), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor type 22) (Lymphocytes)), MYH14 (myosin, heavy chain 14, nonmuscular), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonin deficiency)), SELPLG (selectin P ligand), AOC3 (amineoxidase, copper) Containing 3 (vascular adhesion protein 1)), CTSL1 (catepsin L1), PCNA (proliferating cell nuclear antigen), IGF2 (insulin-like growth factor 2 (somatomedin A)), ITGB1 (integrin, β1 (fibronectin receptor, βpolypeptide,) Antigen CD29 includes MDF2, MSK12)), CAST (carpastatin), CXCL12 (chemokine (CXX motif) ligand 12 (stroma cell-derived factor 1)), IGHE (immunoglobulin heavy chain constant ε), KCNE1 (Potato Potato Open Channel, Isk Related Family, Member 1), TFRC (Transferin Receptor (p90, CD71)), COL1A1 (Collagen, Type I, α1), COL1A2 (Collagen, Type I, α2), IL2RB (Inter) Leukin 2 receptor, β), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoetin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antibacterial peptide) ), PTPN11 (protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solute transport carrier family 2 (promoting glucose transporter), member 1), IL2RA (interroy Kin 2 receptor, α), CCL5 (chemocaine (CC motif) ligand 5), IRF1 (interferon regulator 1), CFLAR (CASP8 and FADD-like apoptosis regulator), CALCA (calcitonin-related polypeptide α), EIF4E (Eukaryotic translation initiation factor 4E), GSTP1 (glutathione S-transferase π1), JAK2 (Yanus kinase 2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (hepalan sulfate proteoglycan 2) , CCL3 (chemokine (CC motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenalinergic) , Β, receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member 2), MMP8 (matrix metallopeptidase 8 (neutrophil colagenase)), NPR2 (sodium diuretic peptide receptor B / guanyl) Acid cyclase B (atrial sodium diuretic peptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS (glutamyl-prolyl-tRNA synthetase), PPARGC1A (peroxysome growth factor activating receptor γ, coactivator 1α), F12 (coagulation factor XII (Hagemann factor)), PECAM1 (platelet / endothelial cell adhesion molecule), CCL4 (chemocaine (CC motif) ligand 4), SERPINA3 (selpin peptidase inhibitor, clade A (α-1 antipeptidease) , Antitrypsin), Member 3), CASR (Calcium Sensing Receptor), GJA5 (Gap-Binding Protein, α5, 40 kDa), FABP2 (Fatigue-Binding Protein 2, Intestine), TTF2 (Transcribing Termination Factor, RNA Peptide II), PROS1 (Protein S (α)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan, β (43 kDa dystrophin-related glycoprotein)), YME1L1 (YME1-like 1 (S. S. cerevisiae)), CAMP (catericidine antibacterial peptide), ZC3H12A (12A containing zinc finger CCCH type), AKR1B1 (ald-ketreductase family 1, member B1 (aldose reductase)), DES (desmin), MMP7 ( Matrix metallopeptidase 7 (matrilysin, uterus)), AHR (aryl hydrocarbon receptor), CSF1 (colony stimulator 1 (macrophages)), HDAC9 (histon deacetylase 9), CTGF (binding tissue growth factor), KCNMA1 (Large Conductance Calcium Activated Potassium Channel, Subfamily M, α Member 1), UGT1A (UDP Glucronosyl Transtransferase 1 Family, Polypeptide A Complex Genesis), PRKCA (Protein Kinase C, α), COMT (Catecol-β) -Methyltransferase), S100B (S100 calcium-binding protein B), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine receptor D4), CAMK2G (calcium / calmodulin-dependent protein) Kinase IIγ), SLC22A2 (solute transport carrier family 22 (organic cation transporter), member 2), CCL11 (chemokine (CC motif) ligand 11), PGF (B321 placenta growth factor), THPO (thrombopoetin), GP6 ( Glycoprotein VI (platelet)), TACR1 (takikinin receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium potential open channel, Shal-related subfamily, member 1) ), LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1 (platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol) Dehydrogenase 1C (class I), γ polypeptide), ALOX12 (arachidonic acid 12-lipoxygenase), AHSG (α-2-HS-sugar protein), BHMT (betaine-homocysteine methyl transferase), GJA4 (gap binding protein, α4) , 37 kDa), SLC25A4 (solute transporter) Body family 25 (mititritic transport carrier; adenine nucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonic acid 5-lipoxygenase activating protein), NUMA1 (nuclear mitotic protein 1), CYP27B1 (Cytochrome P450, Family 27, Subfamily B, Polypeptide 1), CYSLTR2 (Cistinyl leukotriene receptor 2), SOD3 (Superoxide dismutase 3, extracellular), LTC4S (Leukotriene C4 synthase), UCN (Urocortin), GHRL (Grelin / Obestatin prepropeptide), APOC2 (Apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (10 containing Kerhi repeat and BTB (POZ) domains), TNC (Teneisin C), TYMS (thymidylate synthetase), SHCl (SHC (containing 2 domains of Src homology) transforming protein 1), LRP1 (low density lipoprotein receptor-related protein 1), SOCS3 (suppressor 3 for cytokine signaling), ADH1B (alcohol) Dehydrogenase 1B (class I), β-polypeptide), KLK3 (calicrane-related peptidase 3), HSD11B1 (hydroxysteroid (11-β) dehydrogenase 1), VKORC1 (vitamin K epoxy drectinase complex, subunit 1), SERPINB2 (selpin) Peptidase inhibitor, clade B (ovoalbumin), member 2), TNS1 (tensin 1), RNF19A (ring finger protein 19A), EPOR (erythropoetin receptor), ITGAM (integrin, αM (complementary component 3 receptor 3 sub) Unit)), PITX2 (Paired-like homeodomain 2), MAPK7 (Mightogen-activated protein kinase 7), FCGR3A (IgG Fc fragment, low affinity 111a, receptor (CD16a)), LEPR (Leptin receptor), ENG (Endogrin), GPX1 (glutathione peroxidase 1), GOT2 (glutamate oxaloacetate transaminase 2, mitochondria (aspartate aminotransferase 2)), HRH1 (histamine receptor H1), NR112 (nuclear receptor subfamily 1, group I) , Member 2), CRH Tycotropin-releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (potential-dependent anion channel 1), HPSE (heparanase), SFTPD (surfactant protein D), TAP2 (transporter 2, ATP binding cassette) , Subfamily B (MDR / TAP)), RNF123 (ring finger protein 123), PTK2B (PTK2B protein tyrosine kinase 2β), NTRK2 (neurotrophic tyrosine kinase, receptor type 2), IL6R (interleukin 6 receptor) , ACHE (acetylcholine esterase (Yt blood type)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD (P) H dehydrogenase, quinone 1), NR5A1 (Nuclear Receptor Subfamily 5, Group A, Member 1), GJB2 (Gap Binding Protein, β2, 26 kDa), SLC9A1 (Solute Transport Carrier Family 9 (Sodium / Hydrogen Exchanger), Member 1), MAOA (Monoamine Oxide A), PCSK9 (proprotein convertase subtilicin / kexin type 9), FCGR2A (IgG Fc fragment, low affinity IIa, receptor (CD32)), SERPINF1 (selpin peptidase inhibitor, clade F (α-2 anti-plasmin) , Pigment epithelial-derived factor), member 1), EDN3 (endoserine 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest specific 6), SMPD1 (sphingoeline phosphodiesterase 1, acidic lysosome), UCP2 (deconjugated protein 2) (Military, proton carrier)), TFAP2A (transcription factor AP-2α (activated enhancer binding protein 2α)), C4BPA (complementary component 4-binding protein, α), SERPINF2 (selpin peptidase inhibitor, clade F (α-2) Antiplasmin, pigment epithelium-derived factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placenta (Regan isozyme)), CXCR2 (chemokine (CXC motif) receptor 2), SLC39A3 (Solute Transport Carrier Family 39 (Zinc Transporter), Member 3), ABCG2 (ATP Binding Cassette, Subfamily G (WHITE), Member 2), AD A (adenosine deaminase), JAK3 (yanus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), F11 (coagulation factor XI), ATP7A (ATPase, Cu ++ transport, α-polypeptide), CR1 (complementary component (3b / 4b) receptor 1 (Knops blood type)), GFAP (glial fibrous acidic protein), ROCK1 (Rho-related, coiled coil-containing protein kinase) 1), MECP2 (methyl CpG binding protein 2 (let syndrome)), MYLK (myosin light chain kinase), BCHE (butyrylcholine esterase), LIPE (lipase, hormone sensitivity), PRDX5 (peroxyredoxin 5), ADORA1 (adenosin) A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (chemokine (CXC motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, γ2) ), MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CHGA (chromogranin A (parathyroid secretory protein 1)), IAPP (pancreatic islet amyloid polypeptide), RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase / phosphodiesterase 1) , PTHLH (parathyroid hormone-like hormone), NRG1 (neuregulin 1), VEGFC (vascular endothelial growth factor C), ENPEP (glutamylaminopeptidase (aminopeptidase A)), CEBPB (CCAAT / enhancer binding protein (C / EBP)) , Β), NAGLU (N-acetylglucosaminidase, α-), F2RL3 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1), BDKRB1 (bradikinase receptor) B1), ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif, 13), ELANE (elastase, neutrophil expression), ENPP2 (ectonucleotide pyrophosphatase / phosphodiesterase 2), CISH (cytocytosis-induced SH2-containing protein) , GAST (gastrin), MYOC (myocilin, trabecular meshwork-induced kinase response), ATP1A2 (ATPase, Na + / K + transport) , Α2 polypeptide), NF1 (neurofibromin 1), GJB1 (gap binding protein, β1, 32 kDa), MEF2A (muscle cell enhancer factor 2A), VCL (vincurin), BMPR2 (bone-forming protein receptor, type II () Serine / threonine kinase)), TUBB (tubulin, β), CDC42 (cell division cycle 42 (GTP binding protein, 25 kDa)), KRT18 (keratin 18), HSF1 (heat shock transcription factor 1), MYB (v-myb) Myeloblastopathy virus cancer gene homologue (tri)), PRKAA2 (protein kinase, AMP activation, α2 catalytic subunit), ROCK2 (Rho-related, coiled coil-containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein) Related coagulation inhibitors)), PRKG1 (protein kinase, cGMP-dependent, type I), BMP2 (bone-forming protein 2), CTNND1 (catenin (cadherin-related protein), δ1), CTH (cystathionase (cystathionine γ-riase)) , CTSS (catepsin S), VAV2 (vav2 guanine nucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S) -Transtransferase α1), PPIA (peptidyl prolyl isomerase A (cyclophyllin A)), APOH (apolypoprotein H (β-2-glycoprotein I)), S100A8 (S100 calcium-binding protein A8), IL11 (interleukin 11), ALOX15 (arachidonic acid 15-lipoxygenase), FBRN1 (fiburin 1), NR1H3 (nuclear receptor subfamily 1, group H, member 3), SCD (stearoyl-CoA desaturase (Δ-9-desaturase)), GIP (gastric) Inhibitory polypeptide), CHGB (chromogranin B (secret granin 1)), PRKCB (protein kinase C, β), SRD5A1 (steroid-5-alpha-reductase, α-polypeptide 1 (3-oxo-5α-steroid Δ4-) Dehydrogenase α1)), HSD11B2 (hydroxysteroid (11-β) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-α-D-gala) Kusamine: Polypeptide N-Acetylgalactosaminyl transferase 2 (GalNAc-T2)), ANGPTL4 (Angiopoetin-like 4), KCNN4 (Potato intermediate / small conductance calcium activation channel, Subfamily N, Member 4), PIK3C2A (Phonoinocitide-3-kinase, class 2, α-polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major tissue compatible gene) Complex, Class II, DR β 5), BNIP3 (BCL2 / adenovirus E1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4) regulator), S100A12 (S100 calcium binding protein A12), PADI4 (peptidylarginine) Deminase, type IV), HSPA14 (heat shock 70 kDa protein 14), CXCR1 (chemokine (CXC motif) receptor 1), H19 (H19, imprinted maternal expression transcript (non-protein code)), KRTAP19 -3 (keratin-related protein 19-3), IDDM2 (insulin-dependent intrinsic diabetes 2)
, RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, low molecular weight GTP binding protein Rac2)), RYR1 (lianodin receptor 1 (skeleton)), CLOCK (clock homologue (mouse)), NGFR (nerve growth factor receptor) (TNFR superfamily, member 16)), DBH (dopamine β-hydroxylase (dopamine β-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, α4), CACNA1C (calcium channel, potential dependent, L) Type, α1C subunit), PRKAG2 (protein kinase, AMP activation, γ2 non-catalyzed subunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subunit) Family 1, Group H, Member 2), TEK (TEK tyrosine kinase, endothelium), VEGFB (vascular endothelial growth factor B), MEF2C (muscle cell enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2) , TNFRSF11A (tumor necrosis factor receptor superfamily, member 11a, NFKB activator), HSPA9 (heat shock 70 kDa protein 9 (mortarin)), CYSLTR1 (cystenyl leukotriene receptor 1), MAT1A (methionine adenosyltransferase I, α), OPRL1 (opiate receptor-like 1), IMPA1 (inositol (myo) -1 (or 4) -monophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (prosome) , Macropine (macropine) receptor, α type, 6), PSMB8 (proteasome (prosome, macropine) receptor, β type, 8 (large multifunctional peptidase 7)), CHI3L1 (kitinase 3-like 1 (chocolate sugar) Protein-39)), ALDH1B1 (aldehyde dehydrogenase 1 family, member B1), PARP2 (poly (ADP-ribose) polymerase 2), STAR (steroid-producing acute regulatory protein), LBP (lipopolysaccharide binding protein), ABCC6 (ATP binding) Cassette, subfamily C (CFTR / MRP), member 6), RGS2 (regulator of G protein signaling 2, 24k Da), EFNB2 (Ephrin-B2), GJB6 (Gap binding protein, β6, 30 kDa), APOA2 (Apolipoprotein A-II), AMPD1 (Adenosine monophosphate desaturase 1), DYSF (Disferin, limb band type muscle dystrophy 2B) Autosomal recessive inheritance)), FDFT1 (farnesyl diphosphate farnesyl transferase 1), EDN2 (endoserine 2), CCR6 (chemocaine (CC motif) receptor 6), GJB3 (gap binding protein, β3, 31 kDa), IL1RL1 (Interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Valde-Bedle syndrome 4), CELSR2 (cadherin, EGF LAG 7 transmembrane G-type receptor 2 (flamingo homolog) , Drosophila)), F11R (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronoglucosaminidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant) Protein 1 homolog (yeast)), ATF6 (activated transcription factor 6), KHK (ketohexokinase (fructokinase)), SAT1 (spermidine / spermin N1-acetyltransferase 1), GGH (γ-glutamylhydrolase (conjugase, conjugase,) Holylpoly γ-glutamylhydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solute transport carrier family 4, sodium / hydrogen carbonate ion cotransporter, member 4), PDE2A (phosphodiesterase 2A, cGMP stimulant), PDE3B ( Phosphodiesterase 3B, cGMP inhibitory), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin β4, X chain), TXNIP (thioredoxin interacting protein), LIMS1 (LIM and aging cell antigen-like domain 1) , RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), FOXO1 (forkhead box O1), PNPLA2 (patatin-like phosphoripase domain-containing 2), TRH (thyrotropin-releasing hormone), GJC1 (gap binding) Protein, γ1, 45 kDa), SLC17A5 (solute transporter) Body family 17 (anion / sugar transporter), member 5), FTO (body fat mass and obesity-related), GJD2 (gap binding protein, δ2, 36 kDa), PSRC1 (proline / serine-rich coiled coil 1), CASP12 (caspase 12) (Gene / Pseudogene)), GPBAR1 (G protein-conjugated bile acid receptor 1), PXK (PX domain-containing serine / threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles) homolog 1 (Drosophila) (Drosophila))), PBX4 (pre-B cell leukemia homeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1), 15-Sep (15 kDa serenoprotein), CILP2 (chondral middle layer protein 2), TERC (telomerase) RNA components), GGT2 (γ-glutamyl kinase 2), MT-CO1 (mitochrome c oxidase I encoded in mitochondria), and UOX (uric acid oxidase, pseudogene) can be included. Any of these sequences can be the target of the CRISPR-Cas system, for example to address mutations.

In a further embodiment, the chromosomal sequences are Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE (apolipoprotein E), ApoB-100 (apolipoprotein B-100), APOA (apolipoprotein (a)). ), APOA1 (apolipoprotein A1), CBS (cystatione B-synthase), glycoprotein IIb / IIb, MTHRF (5,10-methylenetetrahydrofolate reductase (NADPH), and combinations thereof. In one iteration, the chromosomal sequences involved in cardiovascular disease and the proteins encoded by the chromosomal sequences are targets of the CRISPR-Cas system, Cacna1C, Sod1, Pten, Ppar (α), ApoE, leptin, and theirs. Can be selected from combinations.

Secretase Disorders US Patent Application Publication No. 20110023146 describes genetic modification of cells, animals and proteins associated with secretase-related disorders using zinc finger nucleases. Secretase is essential for processing preproteins into their biologically active forms. Defects in various components of the secretase pathway contribute to many disorders, especially those with amyloid formation or amyloid plaques, which are prominent features such as Alzheimer's disease (AD).

Secretase disorders and the proteins associated with those disorders are a diverse collection of proteins that result in susceptibility to numerous disorders, the presence of the disorder, the severity of the disorder, or any combination thereof. The present disclosure includes editing any chromosomal sequence that encodes a protein associated with secretase disorders. Proteins associated with secretase disorder are typically selected based on the experimental association between the secretase-related protein and the development of secretase disorder. For example, a population with a secretase disorder may have an increased or decreased production rate or circulating concentration of proteins associated with the secretase disorder as compared to a population without a secretase disorder. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with secretase disorders are, but not limited to, those proteins using genomics techniques including DNA microarray analysis, gene expression chain analysis (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). It can be identified by obtaining the gene expression profile of the gene encoding.

As a non-limiting example, proteins associated with secretase disorders include PSENEN (presenilin enhancer 2 homologue (C. elegans)), CTSB (catepsin B), PSEN1 (presenilin 1), APP (amyloid β). (A4) precursor protein), APH1B (anteriopharyngeal insufficiency 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer's disease 4)), BACE1 (β-site APP cleaving enzyme 1), ITM2B (Endogenous membrane protein 2B), CTSD (catepsin D), NOTCH1 (Notch homolog 1, translocation-related (Drosophila)), TNF (tumor necrosis factor (TNF superfamily, member 2)), INS (insulin) ), DYT10 (distony 10), ADAM17 (ADAM metallopeptidase domain 17), APOE (apolypoprotein E), ACE (angiotensin I converting enzyme (peptidyldipeptidase A) 1), STN (statin), TP53 (tumor protein p53) , IL6 (Interleukin 6 (Interferon, β2)), NGFR (Neural Growth Factor Receptor (TNFR Superfamily, Member 16)), IL1B (Interleukin 1, β), ACHE (Acetylcholine Esterase (Yt Blood Type)), CTNNNB1 (catenin (cadherin-related protein), β1, 88 kDa), IGF1 (insulin-like growth factor 1 (somatomedin C)), IFNG (interferon, γ), NRG1 (newregulin 1), CSP3 (caspase 3, apoptosis-related cysteine peptidase) ), MAPK1 (mitogen activated protein kinase 1), CDH1 (cadherin 1, type 1, E-cadherin (epithelial)), APBB1 (amyloid β (A4) precursor protein binding, family B, member 1 (Fe65)), HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase), CREB1 (cAMP response element binding protein 1), PTGS2 (prosenilin endoperoxide synthase 2 (prosenilin G / H synthase and cyclooxygenase)), HES1 (Hairy and Enhancer of Split 1, (Drosophila)), CAT (catalase), TGFB1 (transformed growth factor) Child, β1), ENO2 (enolase 2 (γ, neuron)), ERBB4 (v-erb-a erythroblastic leukemia viral cancer gene homolog 4 (tri)), TRAPPC10 (transport protein particle complex 10), MAOB (Monoamine oxidase B), NGF (nerve growth factor (β-polypeptide)), MMP12 (matrix metallopeptidase 12 (macrophages elastase)), JAG1 (jagd 1 (Arazil syndrome)), CD40LG (CD40 ligand), PPARG (peroxysome proliferation Factor activated receptor γ), FGF2 (fibroblast growth factor 2 (basic)), IL3 (interleukin 3 (colony stimulator, multiplex)), LRP1 (low density lipoprotein receptor-related protein 1), NOTCH4 (Notch Homolog 4 (Drosophila)), MAPK8 (Mightgen Activated Protein Kinase 8), PREP (Prolyl Endopeptidase), NOTCH3 (Notch Homolog 3 (Drosophila)), PRNP (Prion Protein) , CTSG (catepsin G), EGF (epithelial growth factor (β-urogastron)), REN (renin), CD44 (CD44 molecule (Indian blood type)), SELP (selectin P (granular protein 140 kDa, antigen CD62)) , GHR (growth hormone receptor), ADCYAP1 (adenylate cyclase activated polypeptide 1 (pituitary)), INSR (insulin receptor), GFAP (glial fibrous acidic protein), MMP3 (matrix metallopeptidase 3 (stromerai)) Syn 1, proseratinase)), MAPK10 (mitogen-activated protein kinase 10), SP1 (Sp1 transcription factor), MYC (v-myc myeloid disease viral cancer gene homologue (tri)), CTSE (catepsin E), PPARA (Peroxysome growth factor activated receptor α), JUN (jun cancer gene), TIMP1 (TIMP metallopeptidase inhibitor 1), IL5 (interleukin 5 (colony stimulator, eosinophil)), IL1A (interleukin 1, α), MMP9 (matrix metallopeptidase 9 (zelatinase B, 92 kDa geratinase, 92 kDa type IV collagenase)), HTR4 (5-hydroxytryptamine (serotonin) receptor 4), HSPG2 (heparan sulfate) Roteoglycan 2), KRAS (v-Ki-ras2 cursten rat sarcoma virus oncogene homologue), CYCS (cytochrome c, somatic), SMG1 (SMG1 homologue, phosphatidylinositol 3-kinase-related kinase (C.I. Elegance)), IL1R1 (interleukin 1 receptor, type I), PROK1 (prokineticin 1), MAPK3 (mitogen-activated protein kinase 3), NTRK1 (neurotrophic tyrosine kinase, receptor, type 1) , IL13 (Interleukin 13), MME (Membrane Metalloendopeptidase), TKT (Transketrase), CXCR2 (Chemocaine (CXX motif) receptor 2), IGF1R (Insulin-like growth factor 1 receptor), RARA (retinoic acid receptor, α), CREBBP (CREB binding protein), PTGS1 (prostaglandin endoperoxide synthase 1 (prostaglandin G / H synthase and cyclooxygenase)), GALT (galactose-1-phosphate uridylyl) Transtransferase), CHRM1 (cholinergic receptor, muscarinergic 1), ATXN1 (ataxin 1), PAWR (PRKC, apoptosis, WT1, regulator), NOTCH2 (Notch homolog 2 (Drosophila)), M6PR (Mannose-6-phosphate receptor (cation-dependent)), CYP46A1 (cytochrome P450, family 46, subfamily A, polypeptide 1), CSNK1 D (casein kinase 1, δ), MAPK14 (mitogen-activated protein kinase) 14), PRG2 (proteoglycan 2, bone marrow (natural killer cell activator, eosinophil granule major basic protein)), PRKCA (protein kinase C, α), L1 CAM (L1 cell adhesion molecule), CD40 (CD40 molecule, TNF) Receptor superfamily member 5), NR1I2 (nuclear receptor subfamily 1, group I, member 2), JAG2 (jagd 2), CTNND1 (catenin (cadherin-related protein), δ1), CDH2 (cadherin 2, type 1) , N-cadherin (nerve type)), CMA1 (chimase 1, mast cell), SORT1 (soltilin 1), DLK1 (δ-like 1 homolog (Drosophila)), THEM4 (thioesterase superfamily member 4), JUP (binding placoglobin), CD46 (CD46 molecule, receptor regulatory protein), CCL11 (chemokine (CC motif) ligand 11), CAV3 (caveolin 3), RNASE3 (ribonuc) Lease, RNase A family, 3 (eosinophil-cationic protein)), HSPA8 (heat shock 70 kDa protein 8), CASP9 (caspase 9, apoptosis-related cysteine peptidase), CYP3A4 (cytochrome P450, family 3, subfamily A, Polypeptide 4), CCR3 (chemokine (CC motif) receptor 3), TFAP2A (transcription factor AP-2α (activated enhancer binding protein 2α)), SCP2 (sterol carrier protein 2), CDK4 (cycline-dependent kinase) 4), HIF1A (hypoxic inducer 1, α subunit (basic helix-loop-helix transcription factor)), TCF7L2 (transcription factor 7-like 2 (T cell-specific, HMG box)), IL1R2 (interleukin 1) Receptor, type II), B3GALTL (β 1,3-galactosyltransferase-like), MDM2 (Mdm2 p53 binding protein homolog (mouse)), RELA (v-rel reticular endothelial disease virus cancer gene homolog A (tri)), CASP7 (caspase 7, apoptosis-related cysteine peptidase), IDE (insulin degrading enzyme), FABP4 (fatty acid binding protein 4, fat cells), CASK (calcium / carmodulin-dependent serine protein kinase (MAGUK family)), ADCYAP1R1 (adenylate cyclase) Activated polypeptide 1 (pituitary) receptor type I), ATF4 (activated transcription factor 4 (tax responsive enhancer element B67)), PDGFA (platelet-derived growth factor α-polypeptide), C21 or f33 (chromosomal 21 open) Leading frame 33), SCG5 (secret granin V (7B2 protein)), RNF123 (ring finger protein 123), NFKB1 (nuclear factor 1 of B intracellular kappa light chain polypeptide gene enhancer), ERBB2 (v-erb-) b2 erythroblastic leukemia virus cancer gene homolog 2, nerve / glioblastoma-derived cancer gene homolog (bird)), CAV1 (caveolin 1, caveora protein, 22 kDa), MMP7 (matrix metallopeptidase 7 (matrilysin, uterus)), TGFA (transformed growth factor, α), RXRA (retinoid X receptor, α), STX1A (synthaxin 1A (brain)), PSMC4 (proteasome (prosome, macropine) 26S subunit, ATP Aze, 4), P2RY2 (purine receptor P2Y, G protein conjugate, 2), TNFRSF21 (tumor necrosis factor receptor superfamily, member 21), DLG1 (disc, large homologue 1 (Drosophila)), NUMBL (Numb homolog (Fruit fly (Drosophila)), SPN (sialophorin), PLSCR1 (phospholipid scramblerase 1), UBQLN2 (ubiquilin 2), UBQLN1 (ubiquilin 1), PCSK7 (proprotein convertase subtilisin / kexin type 7 ), SPON1 (Spondin 1, extracellular matrix protein), SILV (Silver Homolog (mouse)), QPCT (Glutaminyl peptide cyclotransferase), HESS (Hairy and Enhancer of Split 5 (Fruitfly)), Includes GCC1 (contains GRIP and coiled coil domains 1), and any combination thereof.

Animals or cells undergoing genetic modification have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disrupted chromosomal sequences and disruptions encoding proteins associated with secretase disorder. It may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosome-integrated sequences encoding the disrupted secretase disorder-related protein.

Liver or hepatocyte targeting; hemophilia hepatocyte targeting is provided. It may be in vitro or in vivo. Hepatocytes are preferred. Delivery of the CRISPR protein can be by viral vector, particularly AAV (and specifically AAV2 / 6) vector. These can be administered by intravenous injection.

The preferred target for the liver is the albumin gene, whether in vitro or in vivo. This is a so-called "safe harbor" because albumin is expressed at extremely high levels and thus allows some reduction in albumin production after successful gene editing. Also, the high levels of expression seen from the albumin promoter / enhancer can produce useful levels of modification or transgene production (from the inserted donor template) even if only a few hepatocytes have been edited. It is preferable to realize it.

Albumin introns 1 are described in Wechsler et al. (Reported in the 57th Annual Meeting and Expotion of the American Society of Hematology-Abstracts are available at https://ash.confex.com/ash.confex.com/ash/2015/web (Announced today) shows that it is a suitable target site. Their study used Zn fingers to cleave DNA at this target site, creating suitable guide sequences that guide cleavage by the CRISPR protein at the same site.

Using targets within highly expressed genes such as albumin (genes with highly active enhancers / promoters), Wechsler et al. As reported by, it has also become possible to use promoterless donor templates, which are also widely applicable beyond liver targeting. Other examples of highly expressed genes are known.

Liver-related blood disorders, especially hemophilia and more specifically hemophilia B
We have shown that successful gene editing of hepatsites has been achieved in mice (both in vitro and in vivo) and non-human primates (in vivo), and that gene editing / genome engineering treatment of hepatocytes in hepatocytes is feasible. There is. In particular, expression of the human F9 (hF9) gene in hepatocytes has been shown in non-human primates, suggesting treatment for human hemophilia B (hemophilia B).

Wechsler et al. At the 57th Annual Meeting and Expotion of the American Society of Hematology (Abstract was published on December 6, 2015, at https://ash.confex.com/ash.comfex.com/ash.com/up. (Possible), we reported that we succeeded in expressing human F9 (hF9) from hepatocytes by in vivo gene editing in non-human primates. This was achieved using 1) two zinc finger nucleases (ZFNs) targeting intron 1 at the albumin locus, and 2) a human F9 donor template construct. ZFN and donor templates are encoded by separate hepatic adeno-associated virus serotype 2/6 (AAV2 / 6) vectors, and intravenous injection of these vectors results in a modified copy of the hF9 gene in a proportion of liver hepatocytes. Was targeted and inserted at the albumin locus.

The albumin locus was selected as the "safe harbor" because the production of this most abundant plasma protein exceeds 10 g / day and a modest reduction in its level is well tolerated. Genome-edited hepatocytes were driven by a highly active albumin enhancer / promoter to produce a therapeutic amount of normal hFIX (hF9) rather than albumin. Target integration of the HF9 trans gene at the albumin locus and splicing of this gene onto albumin transcripts were shown.

Mouse test: C57BL / 6 mice administered by tail intravenous injection with vehicle (n = 20) or AAV2 / 6 vector (n = 25) encoding mouse surrogate reagent at 1.0 × 10 ¹³ vector genome (vg) / kg Was done. Elisa analysis of plasma hFIX in treated mice showed a peak level of 50-1053 ng / mL, which persisted for a 6-month study period. Analysis of FIX activity from mouse plasma confirmed biological activity according to the expression level.

Non-human primate (NHP) study: AAV2 / 6 vector encoding an NHP-targeted albumin-specific ZFN and a human F9 donor given a single intravenous co-injection at 1.2 × 10 ¹³ vg / kg (n = 5 /) Group),> 50 ng / mL (normal> 1%) was obtained with this large animal model. With higher AAV 2/6 doses (up to 1.5 x 10 ¹⁴ vg / kg), up to 1000 ng / ml (or 20% of normal) in some animals over the study period (3 months). Plasma hFIX levels were produced, and up to 2000 ng / ml (or 50% of normal) in one animal.

Treatment was well tolerated in mice and NHP, and there were no significant toxicological findings associated with AAV2 / 6 ZFN + donor treatment at therapeutic doses in either species. Sangamo (CA, USA) has since filed and approved the FDA for permission to conduct the world's first human clinical trial for in vivo genome editing applications. This follows the approval of EMEA for the treatment of Glybera gene therapy for lipoprotein lipase deficiency.

Therefore, in some embodiments, it is preferable to use some or all of the following:
Administered by AAV (particularly AAV2 / 6) vector, preferably intravenous injection;
Albumin as a target for gene editing / insertion of transgenes / templates-especially in intron 1 of albumin;
-Human F9 donor template; and / or-promoterless donor template.

Hemophilia B
Therefore, in some embodiments, the present invention is preferably used in the treatment of hemophilia B. Therefore, it is preferred that a template be provided and that it is the human F9 gene. It will be appreciated that the hF9 template contains wt or the "correct" version of hF9 for therapeutic efficacy.

In an alternative embodiment, the hemophilia B version of F9 may be delivered to produce a model organism, cell or cell line (eg, mouse or non-human primate model organism, cell or cell line), which model organism. , Cells or cell lines have or possess the hemophilia B phenotype, i.e., do not have the ability to produce wt F9.

Hemophilia A
In some embodiments, the F9 (factor IX) gene may be replaced with the F8 (factor VIII) gene described above for the treatment of hemophilia A (by providing the correct F8 gene) and /. Or it leads to the creation of a hemophilia A model organism, cell or cell line (by providing the F8 gene for the incorrect hemophilia A version).

Hemophilia C
In some embodiments, the F9 (factor IX) gene may be replaced with the F11 (factor X I) gene described above for the treatment of hemophilia C (by providing the correct F11 gene) and / Or leads to the creation of a hemophilia C model organism, cell or cell line (by providing the F11 gene for the incorrect hemophilia C version).

Other pathologies Cystic fibrosis (CF)
In some embodiments, treatment, prevention or diagnosis of cystic fibrosis is provided. The target is preferably the SCNN1A or CFTR gene. This is described in Pamphlet International Publication No. 2015157070, the disclosure of which is incorporated by reference herein.

Cancer and CAR-T
In some embodiments, treatment, prevention or diagnosis of cystic fibrosis is provided. The target is preferably one or more of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC or TRBC genes. Cancers include lymphoma, chronic lymphocytic leukemia (CLL), B-cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma (NHL), and diffuse large cell lymphoma. (DLCL), multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colonic rectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, stellate It may be one or more of cell carcinoma, mesenteric tumor, head and neck cancer, and myelopathy. This can be achieved with engineered chimeric antigen receptor (CAR) T cells. This is described in Pamphlet International Publication No. 2015161276, the disclosure of which is incorporated by reference herein.

Herpes simplex virus types 1 and 2 In some embodiments, treatment, prevention or diagnosis of HSV-1 (herpes simplex virus type 1) is provided. The target is preferably the UL19, UL30, UL48 or UL50 genes of HSV-1. This is described in Pamphlet International Publication No. 2015153789, the disclosure of which is incorporated by reference herein.

In other embodiments, treatment, prevention or diagnosis of HSV-2 (herpes simplex virus type 2) is provided. The target is preferably the UL19, UL30, UL48 or UL50 genes of HSV-2. This is described in Pamphlet International Publication No. 2015153791, the disclosure of which is incorporated by reference herein.

The present invention relates to the delivery of the CRISPR protein complex and the use of RNA-guided endonucleases in cells and organisms, as set forth in the following articles, which are incorporated herein by reference herein. It can be further exemplified and extended based on aspects of development and use of CRISPR-Cas9:
-"Multiplex genome engineering using CRISPR / Cas systems (CRISPR / Cas systems)". Kong, L. , Ran, F. A. , Cox, D. , Lin, S.A. , Barretto, R.M. , Hubib, N. et al. , Hsu, P. et al. D. , Wu, X. , Jiang, W. et al. , Marraffini, L. et al. A. , & Zhang, F. Science Feb 15; 339 (6121): 819-23 (2013);
-RNA-guided editing of the bacterial genome using the CRISPR-Cas system (RNA-guided editing of bacterial genomes CRISPR-Cas systems). Jiang W. , Bikerd D. , Cox D. , Zhang F, Marraffini LA. Nat Biotechnol Mar; 31 (3): 233-9 (2013);
"One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR / Cas-Mediated Genome Engineering" in mice with mutations in multiple genes. Wang H. , Yang H. , Shivalila CS. , Dawraty MM. , Cheng AW. , Zhang F. , Jaenich R.M. Cell May 9; 153 (4): 910-8 (2013);
"Optical control of mammal endocenous transcription and epigenetic states". "Mammalian endogenous transcription and epigenetic states". Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Kong L, Platt RJ, Scott DA, Church GM, Zhang F. Nature. Aug 22; 500 (7463): 472-6. doi: 10.1038 / Nature12466. EPub 2013 Aug 23 (2013);
"Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity" with RNA-guided CRISPR Cas9 to enhance genome editing specificity. Ran, FA. , Hsu, PD. , Lin, CY. , Goodenberg, JS. , Konermann, S.A. , Trevino, AE. , Scott, DA. , Inoue, A. , Matoba, S.A. , Zhang, Y. et al. , & Zhang, F. Cell Aug 28. pii: S0092-8674 (13) 01015-5 (2013-A);
"DNA targeting specificity of RNA-guided Cas9 nucleoses". "RNA-guided Cas9 nuclease DNA targeting specificity (DNA targeting specificity of RNA-guided Cas9 nucleoses)". Hsu, P.M. , Scott, D.I. , Weinstein, J. Mol. , Ran, FA. , Konermann, S.A. , Agarwalla, V.I. , Li, Y. , Fine, E.I. , Wu, X. , Salem, O.D. , Cradick, TJ. , Marraffini, LA. , Bao, G.M. , & Zhang, F. Nat Biotechnol doi: 10.1038 / nbt. 2647 (2013);
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Each of these is incorporated herein by reference and may be considered in the practice of the present invention, and is briefly discussed below:
・ Kong et al. Engineering the type II CRISPR / Cas system used in eukaryotic cells based on both Streptococcus thermophilus Cas9 and also Streptococcus pyogenes Cas9, where Cas9 nuclease directs low molecular weight RNA. It was demonstrated that it can induce accurate DNA cleavage in human and mouse cells. The authors' study further showed that Cas9, which is converted to a nicking enzyme, can be used to promote homologous recombination repair in eukaryotic cells with minimal mutagenic activity. In addition, our study demonstrates that encoding multiple guide sequences into a single CRISPR sequence can allow simultaneous editing of several at endogenous genomic locus sites within the mammalian genome. We have demonstrated the easy programmable potential and widespread applicability of RNA-guided nuclease technology. It has thus become possible to use RNA to program sequence-specific DNA breaks in cells, defining a new class of genomic engineering tools. These studies further showed that other CRISPR loci may be transplantable into mammalian cells and may also mediate mammalian genome cleavage. Importantly, it can be envisioned that some aspects of the CRISPR / Cas system can be further improved to increase their efficiency and versatility.
・ Jiang et al. Accurately mutate to the genomes of Streptococcus pneumoniae and Escherichia coli using clustered equidistant short-chain batch repeat (CRISPR) -related Cas9 endonucleases complexed with dual RNA. Was introduced. This technique relies on killing non-mutant cells by dual RNA: Cas9-induced cleavage at the target genomic site, avoiding the need for selectable markers or counterselective systems. This study reported that reprogramming dual RNA: Cas9 specificity by rearranging the sequence of short CRISPR RNA (crRNA) to result in single and multiple nucleotide changes results in template editing. .. This study showed that the simultaneous use of two crRNAs allowed mutagenesis multiplexing. Furthermore, when this technique was used in combination with recombination, in Streptococcus pneumoniae (S. pneumoniae), nearly 100% of the cells recovered using the described technique contained the desired mutation and E. coli (E. coli). In E. coli), 65% of the recovered cells contained mutations.
・ Wang et al. (2013), by using the CRISPR / Cas system, a plurality of conventionally prepared at multiple stages by successive recombination in embryonic stem cells and / or crossing of mice with a time-consuming single mutation. Mice carrying mutations in the gene were created in one step. The CRISPR / Cas system can significantly accelerate in vivo studies of functionally overlapping genes and superior gene interactions.
・ Konermann et al. (2013) is the need for versatile and robust techniques in the art that allow the optical and chemical regulation of DNA binding domains based on the CRISPR Cas9 enzyme and also transcriptional activator-like effectors. Dealed with.
・ Ran et al. (2013-A) described a method of introducing a target double-strand break by combining a Cas9 nickase mutant with a pair of guide RNAs. This is because the Cas9 nuclease of the microbial CRISPR-Cas system is targeted to specific genomic loci by the guide sequence, but the guide sequence can tolerate some mismatch with the DNA target and is therefore undesirable off. Address the issue of being able to promote target mutagenesis. Because individual nicks in the genome are repaired with high fidelity, double-strand breaks require simultaneous nicking with properly offset guide RNAs, and bases that are specifically recognized for target breaks. The number increases. The authors used paired nicking to reduce off-target activity in cell lines by 50 to 1,500 times and promote gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. Demonstrated to get. This versatile strategy enables a wide variety of genome editing applications that require high specificity.
・ Hsu et al. (2013) characterized the specificity of SpCas9 targeting in human cells to signal target site selection and avoid off-target effects. This study evaluated SpCas9-induced indel mutation levels at over 700 guide RNA variants and over 100 predictive genomic off-target loci in 293T and 293FT cells. These authors show that SpCas9 is sensitive to the number, location and distribution of mismatches and tolerates mismatches between guide RNA and target DNA at various positions in a sequence-dependent manner. The authors further showed that SpCas9-mediated cleavage is unaffected by DNA methylation and that doses of SpCas9 and sgRNA can be titrated to minimize off-target modification. In addition, to facilitate mammalian genome engineering applications, the authors reported the provision of web-based software tools to guide target sequence selection and validation as well as off-target analysis.
・ Ran et al. (2013-B) is a set for Cas9-mediated genome editing using non-homologous end joining (NHEJ) or homologous recombination repair (HDR) in mammalian cells and for creating modified cell lines for downstream function studies. The tools of are described. To minimize off-target cleavage, the authors further described a double nicking strategy using a Cas9 nickase mutant containing a pair of guide RNAs. The protocol provided by these authors provided experimental guidance on target site selection, evaluation of cleavage efficiency, and analysis of off-target activity. This study showed that genetic modification can be achieved within just 1-2 weeks, starting with target design, and that modified clonal cell lines can be induced within 2-3 weeks.
-Shalem et al. Described a new way to study gene function on a genome-wide scale. Our study found that delivery of a genome-wide CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences was negative and positive selection in human cells. It was shown that both screenings were possible. First, the authors have shown the identification of genes essential for cell survival in cancer and pluripotent stem cells using the Gecko library. Next, the authors screened genes involved in resistance to the therapeutic agent vemurafenib, whose deficiency inhibits the mutant protein kinase BRAF, in a melanoma model. The authors' study showed that the top-ranked candidates included the previously validated genes NF1 and MED12 as well as the novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high degree of agreement and a high rate of hit confirmation between independent guide RNAs targeting the same gene, thus demonstrating the promise of genome-scale screening with Cas9.
・ Nishimasu et al. Reported the crystal structure of Streptococcus pyogenes Cas9, which complexes with sgRNA and its target DNA at a resolution of 2.5 Å. This structure revealed a two-lobe structure that accepts sgRNA: DNA heteroduplexes in the positively charged groove at the interface between the target recognition lobe and the nuclease lobe. The recognition lobe is critical to the binding of sgRNA and DNA, whereas the nuclease lobe contains the HNH and RuvC nuclease domains, which are located at appropriate positions for cleavage of the complementary and non-complementary strands of the target DNA, respectively. It is in. The nuclease lobe also contains a carboxyl-terminated domain that is involved in the interaction with the protospacer flanking motif (PAM). This high-resolution structural analysis and associated functional analysis will reveal the molecular mechanism of RNA-guided DNA targeting by Cas9, which in turn is paving the way for rational design of new versatile genome editing technologies. ..
・ Wu et al. Mapped the genome-wide binding site of catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with a single guide RNA (sgRNA) in mouse embryonic stem cells (mESC). The authors have found that each of the four sgRNAs tested has dCas9 on dozens to thousands of genomic sites, often characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer flanking motif (PAM). Shown to be targeted. The inaccessibility of chromatin reduces dCas9 binding to other sites containing matching seed sequences; thus 70% of off-target sites associate with the gene. The authors showed that targeted sequencing of 295 dCas9 binding sites in a catalytically active Cas9-transfected mESC identified only one site mutated above background levels. The authors have proposed a two-state model of Cas9 binding and cleavage, in which seed matching causes binding, but cleavage requires extensive pairing with the target DNA.
・ Platt et al. Established Cre-dependent Cas9 knock-in mice. The authors have demonstrated in vivo and exobibo genome editing using adeno-associated virus (AAV) -mediated, lentivirus-mediated, or particle-mediated delivery of guide RNAs in neurons, immune cells, and endothelial cells.
・ Hsu et al. (2014) is a review paper that broadly considers the history of CRISPR-Cas9 from yogurt to genome editing, including gene screening of cells.
・ Wang et al. (2014) relates to a pooled loss-of-function gene screening technique suitable for both positive and negative selections using a genome-wide lentivirus single guide RNA (sgRNA) library.
・ Doench et al. Creates a pool of sgRNAs that tie across all possible target sites in the panel of 6 endogenous mouse genes and 3 endogenous human genes and their ability to produce null alleles of that target gene. Was quantitatively evaluated by antibody counterstaining and flow cytometry. The authors have shown that optimization of PAM enhances activity and also provided an online tool for designing sgRNAs.
・ Switch et al. Demonstrate that AAV-mediated SpCas9 genome editing may enable reverse genetics studies of gene function in the brain.
・ Konermann et al. (2015) discusses the ability to add multiple effector domains, such as transcriptional activators, functions and epigenome regulators, to appropriate locations on guides such as stems or tetraloops with and without linkers.
-Zetsche et al. Demonstrates that the Cas9 enzyme can be split in two and thus can control the assembly of Cas9 for activation.
・ Chen et al. Revolves around multiple screening by demonstrating that genome-wide in vivo CRISPR-Cas9 screening in mice reveals regulatory genes for lung metastases.
・ Ran et al. (2015) demonstrates that SaCas9 and its genome editing capabilities cannot be estimated from biochemical assays. Shalem et al. (2015) describes a method of synthetically suppressing (CRISPRi) or activating (CRISPRa) expression using a fusion of catalytically inactive Cas9 (dCas9), arrayed and pooled screening. It shows advances in using Cas9 for genome-wide screening, including genome-wide screening, knockout techniques that inactivate genomic loci, and strategies for regulating transcriptional activity.
-Shalem et al. (2015) describes a method of synthetically suppressing (CRISPRi) or activating (CRISPRa) expression using a fusion of catalytically inactive Cas9 (dCas9), arrayed and pooled screening. It shows advances in using Cas9 for genome-wide screening, including genome-wide screening, knockout techniques that inactivate genomic loci, and strategies for regulating transcriptional activity.
・ Xu et al. (2015) evaluated DNA sequence features that contribute to the efficiency of single guide RNAs (sgRNAs) in CRISPR-based screening. The authors investigated the efficiency of CRISPR / Cas9 knockout and the nucleotide priority at the cleavage site. The authors also found that the sequence priority of CRISPRi / a was substantially different from that of CRISPR / Cas9 knockout.
・ Parnas et al. (2015) identified a gene that regulates the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS) by introducing the genome-wide pool CRISPR-Cas9 library into dendritic cells (DCs). Known regulators of TLR4 signaling and previously unknown candidates were identified and classified into three functional modules with individual effects on canonical responses to LPS.
Ramana et al (2015) demonstrated cleavage of viral episomal DNA (ccDNA) in infected cells. The HBV genome exists in the nucleus of infected hepatitis as a 3.2 kb double-stranded episomal DNA species called covalently bound closed circular DNA (ccDNA), which is an HBV life cycle in which replication is not inhibited by current therapies. Is the main constituent of. The authors have shown that sgRNAs that specifically target highly conserved regions of HBV robustly suppress viral replication and deplete ccDNA.
・ Nishimasu et al. (2015) describes the crystal structure of SaCas9 in a complex with a single guide RNA (sgRNA) containing 5'-TTGAAT-3'PAM and 5'-TTGGGT-3'PAM and a double-stranded DNA target thereof. reported. Structural comparisons of SaCas9 and SpCas9 revealed both structural conservation and diversity, explaining their characteristic PAM specificity and orthologous sgRNA recognition.
・ Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional study of non-coding genomic elements. The authors developed a pooled CRISPR-Cas9 guide RNA library to perform in-situ saturation mutagenesis of human and mouse BCL11A enhancers, which revealed important features of the enhancers.
-Zetsche et al. (2015) reported the characterization of Cpf1, a class 2 CRISPR nuclease derived from Francisella novicida U112, which has characteristics different from Cas9. Cpf1 is a single RNA-induced endonuclease that lacks tracrRNA, utilizes a T-rich protospacer flanking motif, and cleaves DNA by adherent end-type DNA double-strand breaks.
・ Shmakov et al. (2015) reported three distinctive Class 2 CRISPR-Cas systems. The two CRISPR enzymes (C2c1 and C2c3) contain the RuvC-like endonuclease domain that is remotely associated with Cpf1. Unlike Cpf1, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. The third enzyme (C2c2) contains two expected HEPNRNase domains and is tracrRNA independent.
Slaymaker et al (2016) reported an improvement in the specificity of Streptococcus pyogenes Cas9 (SpCas9) using structurally guided protein engineering. The authors have developed a "specificity-enhanced" SpCas9 (eSpCas9) mutant that maintains robust on-target cleavage while reducing off-target effects.

Also, "Dimeric CRISPR RNA-guided FokI nucleoses for high-high-specific genome editing for highly specific genome editing", Shengdar Q. et al. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. et al. Foden, Visual Thepar, Deepak Reyon, Mathew J. et al. Goodwin, Martin J.M. Aryee, J. et al. Keith Jung Nature Biotechnology 32 (6): 569-77 (2014) relates to a dimeric RNA-guided FokI nuclease that recognizes elongated sequences and is capable of highly efficient editing of endogenous genes in human cells.

For general information about the CRISPR-Cas system, its constituents, and the delivery of such constituents, including their preparation and use, including with respect to methods, materials, delivery vehicles, vectors, particles, AAV, and amounts and formulations. , All useful in the practice of the present invention, US Pat. 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(SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUNCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS); US Provisional Patent Application No. 62 / 055,484, September 25, 2014, CRISPR-CAS SYSTEMS. Systems, methods and compositions for sequence manipulation by system (SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUNCE MANIPULATION WITH OPTIMIZED CRISPR-CAS SYSTEMS) ”; US Provisional Patent Application No. 37/12/87, CRISPR-CAS SYSTEMS. 4th, "Systems, methods and compositions for sequence manipulation by the optimized functional CRISPR-CAS system (SYSTEMS, CRISPR AND COMPOSITIONS FOR CRISPR-CASE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS" US provisional CRISPR-CAS 62 / 054,651, September 24, 2014, "Delivery, Use and Therapeutic Application of CRISPR-CAS Systems and Compositions for Modeling Competition for Multiple Cancer Mutations in vivo (DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS Delivery, use and therapeutic application of the CRISPR-CAS system and composition for modeling competition for cancer mutations in (DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO) ”; US Provisional Patent Application Nos. 62/054,675, September 24, 2014, and September 24, 2014. , June 17, 2015, "Delivery, Use and Therapeutic Application of CRISPR-CAS Systems and Compositions in Nerve Cells / Tissues (DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS INNURESON)" US Provisional Patent Application No. 62 / 054,528, September 24, 2014, "delivery, use and therapeutic application of CRISPR-CAS systems and compositions in immune disorders or disorders (DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF). THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISASEASES OR DISORDERS) ”; US Provisional Patent Application No. 62 / 055,454, September 25, 2014,“ Disorders and Diseases Using Cell Permeable Peptide (CPP) CRISPR-CAS system and composition for delivery, use and therapeutic application (DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDEST US Provisional Patent Application No. 62 / 055,460, September 25, 2014, "Multifunctional CRISPR Complex and / or Functional CRISPR Complex with Optimized Enzymes Linked (MULTIFUNCTIONAL-CRISPR COMPLEXES AND / OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES) " US Provisional Patent Application No. 62 / 087,475, Dec. 4, 2014 and No. 62 / 181,690, June 18, 2015, "Optimized Functionality CRISPR- Functional screening by CAS system (FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS); US Provisional Patent Application No. 62 / 055,487, September 25, 2014, "Optimized Functional CRISPR-CAS" System-based functional screening (FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS); US provisional patent application No. 62 / 087,546, December 4, 2014 and No. 62 / 181,687, 2015. June 18, 2014, "Multifunctional CRISPR complex and / or functional CRISPR complex to which an optimizing enzyme is linked ((MULTIFUNCTIONAL CRISPR COMPLEXES AND / OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES)" Patent Application No. 62 / 098,285, December 30, 2014, also "CRISPR MEDIATED IN VIVO MODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS" Can be mentioned.

US Provisional Patent Application No. 62 / 181,659, June 18, 2015 and No. 62 / 207,318, August 19, 2015, "CAS9 Orthologs and Variants for Sequence Manipulation" Engineering and optimization of systems, methods, enzymes and guide scaffolds (ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FOR SEQUEN ". US Provisional Patent Application No. 62 / 181,663, June 18, 2015 and No. 62 / 245,264, October 22, 2015, "NOVEL CRISPR ENZYMES AND SYSTEMS) ”, US Provisional Patent Application No. 62 / 181,675, June 18, 2015, No. 62 / 285,349, October 22, 2015, No. 62 / 296,522. No., February 17, 2016, and No. 62 / 320,231, April 8, 2016, "NOVEL CRISPR ENZYMES AND SYSTEMS", US Provisional Patent Application No. 62 / 232,067, September 24, 2015, US Patent Application No. 14 / 975,085, October 18, 2015, European Application EP16150428.7, US Provisional Patent Application No. 62 / 205,733, August 16, 2015, US Provisional Patent Application No. 62 / 201,542, August 5, 2015, US Provisional Patent Application No. 62 / 193,507. , 2015, and US Provisional Patent Application No. 62 / 181,739, June 18, 2015, respectively, entitled "NOVEL CRISPR ENZYMES AND SYSTEMS". And US Provisional Patent Application No. 62 / 245,270, October 22, 2015, "NOVEL CRISPR ENZYMES AND SYSTEMS". Also, US Provisional Patent Application No. 61 / 939,256, February 12, 2014, and International Publication No. 2015/089473 Pamphlet (PCT / US2014 / 070152), October 12, 2014, Each title "Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation (ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FOR SEQUENCE MAN" is also listed. Also, PCT / US2015 / 045504, August 15, 2015, US Provisional Patent Application No. 62 / 180,699, June 17, 2015, and US Provisional Patent Application No. 62 / 038,358. The specification, August 17, 2014, also includes the titles "Genome Editing with CAS9 Nickase (GENOME EDITING USING CAS9 NICKASES)", respectively.

Each of these patents, patent gazettes, and applications, as well as all references cited or referenced in them or during their examination (“application citations”) and all references cited or referenced in the application citations. Along with any instructions, instructions, product specifications, and product sheets for any product referred to therein or incorporated herein by reference and incorporated herein by reference. , Which is incorporated herein by reference and can be used in the practice of the present invention. All documents (eg, these patents, patent gazettes and applications, and application citations) are to the same extent that each individual document is considered to be concretely and individually indicated to be incorporated by reference. Incorporated herein by reference.

In addition, a method for preparing sgRNA / Cas9 protein-containing particles, in which a mixture containing sgRNA and Cas9 protein (and optionally an HDR template) is used as a surfactant, phospholipid, biodegradable polymer, lipoprotein and alcohol. Methods involving the steps of including or mixing with a mixture consisting essentially of or consisting of them; and with respect to particles from such methods, "CRISPR-CAS for targeting disorders and diseases with particle delivery components. Delivery, use and therapeutic application of systems and compositions (DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISASES INST. Book, Agent Reference No. 47627.99.2060 and BI-2013 / 107 (US Provisional Patent Application No. 62 / 054,490 filed September 24, 2014; filed June 10, 2014. 62 / 010, 441; and 61 / 915, 118, 61 / 915, 215 and 61, respectively, filed on December 12, 2013. / 915, 148 claiming priority from one or more or all of them) (“Particle Delivery PCT”) (incorporated herein by reference). For example, Cas9 protein and sgRNA are preferably in a sterile nuclease-free buffer, such as 1 x PBS, in a preferred, eg, 3: 1-1: 3 or 2: 1-1: 2, or 1: 1 molar. In proportion, they were mixed together at a suitable temperature, such as 15-30 ° C., such as 20-25 ° C., for example room temperature, for a suitable time, such as 15-45, for example 30 minutes. Apart from that, surfactants such as cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); phospholipids such as dimyristoylphosphatidylcholine (DMPC); biodegradable polymers such as ethylene. Glycolpolymers or PEGs and lipoproteins such as low density lipoproteins such as cholesterol, or particle components containing them, are alcohols, preferably C _1-6 alkyl alcohols such as methanol, ethanol, isopropanol, eg. It was dissolved in 100% ethanol. These two solutions were mixed together to form particles containing the Cas9-sgRNA complex. Therefore, the sgRNA may be pre-formed into a complex with the Cas9 protein before forming the entire complex as particles. Different components of different molar ratios known to facilitate delivery of nucleic acids to cells (eg 1,2-dioleoil-3-trimethylammonium propane (DOTAP), 1,2-ditetradecanoyl-sn- Formulations may be made with glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG), and cholesterol), eg DOTAP: DMPC: PEG: cholesterol molar ratios are DOTAP100, DMPC0, PEG0, cholesterol 0; or DOTAP90, DMPC0, PEG10, cholesterol 0; or DOTAP90, DMPC0, PEG5, cholesterol 5, DOTAP100, DMPC0, PEG0, cholesterol 0. The application therefore includes the step of mixing the sgRNA, the Cas9 protein and the constituents forming the particles; and the particles from such a mixing step. Aspects of the invention are particles; eg, a mixture containing sgRNA and / or Cas9 as in the present invention and a particle-forming component, such as in particle delivery PCT, to form particles. Thus, for example, particles using a method similar to particle delivery PCT and particles from such a mixing step (or, of course, other particles containing sgRNA and / or Cas9 as in the present invention) may be included.

Here, the present invention will be further described by the following non-limiting examples.

The following examples are provided as examples of the inventions described herein. The scientific rationale that motivated this study, described below, should not be construed to limit the claimed subject matter in any way or impose any mechanical or other requirements. It should be understood that it does not.

Scientific rationale Although not desired to be bound by any theory described herein, we intend to create a modified mutant SpCas9 enzyme that exhibits improved target specificity, Streptococcus pyogenes. (Streptococcus pyogenes) We devised a modification strategy for Cas9 (SpCas9). The same rationale can be applied to any Cas9 ortholog. This enhanced specificity can be achieved in mutants that exhibit reduced activity for non-target loci while maintaining adequate activity for the intended target locus. The activity in the assays described below relates to the nuclease activity that appears as a DNA break when measured by the formation of an indel. Such activity appears to be related to the ability of the CRISPR complex (ie, the complex between the Cas9 enzyme and the guide RNA) to bind to relevant sites on DNA. Therefore, a decrease in activity towards a non-target site can be expected to result from a decrease in CRISPR complex binding at that site. Therefore, it can be expected that the modified Cas9 enzyme, which exhibits reduced activity on the non-target locus as compared to the non-modified (eg, wild-type) enzyme, does not bind very well to the non-target site. Nishimasu et al. (Cell, 2014,156 (5), pp935-49) at 2.5 Å resolution of SpCas9 mutant enzyme in a complex comprising a 98 nucleotide long single guide RNA (sgRNA) and a stretch of target DNA containing 23 nucleotides in length. The crystal structure is reported. Based on these structural data, we have identified a positively charged region located between RuvC-III and the HNH domain. We speculated that this groove could accept non-target strands after disruption of normal Watson-Crick base pairing when the Cas9 enzyme bound to the relevant DNA region. Positively charged residues in this region of Cas9 can serve to stabilize the interaction between the enzyme and DNA by interacting with the negatively charged phosphodiester skeleton of the non-target strand of DNA. We hypothesize that the substitution of a positively charged residue of Cas9 can disrupt the interaction with the non-target strand. If this interaction is sufficiently disrupted, proper activity on the target site can be maintained, but the interaction with the guide sequence due to one or more mismatches compared to the non-target site, usually the target sequence. The activity of the enzyme on (which may appear to be weaker) can be reduced. Surprisingly, we have discovered that modification of Cas9 can actually reduce off-target activity. See also FIG. 1 and its discussion herein.

See Kleinstiber BP et al, in addition to the present invention, where substitution of positively charged residues disrupts interactions with the DNA backbone, leading to reduced enzyme activity on non-target sites while maintaining proper activity on target sites. Will be done. The authors describe SpCas9 triple-substituted variants (R661A / Q695A / Q926A) and quadruple-substituted variants (N497A / R661A / Q695A / Q926A) that contain mutations in the REC1 domain. These substitutions involve residues designed to disrupt hydrogen bonds to the DNA phosphate backbone, and this mutant has been reported to have high on-target activity and minimal off-target activity. (Kleinstiber BP et al., "High-fidelity CRISPR-Cas9 nucleic acid with no detectable genome-wide 16 nuclease," which does not have a detectable genome-wide off-target effect. 28; 529 (7587): 490-5. Doi: 10.1038 / nature 16526. Epub 2016 Jan 6.

In addition, we also ensure the stability of the interaction between the enzyme and the non-target strand after disruption of normal Watson-Crick base pairing when the Cas9 enzyme binds to the relevant DNA region. It is also assumed that the amino acid residues of the CRISPR enzyme, which may have an increasing effect, can be modified. For example, replacement of a non-positively charged amino acid residue with a positively charged amino acid in an unmodified enzyme may have the effect of stabilizing the interaction between the enzyme and the non-target chain by increasing the net positive charge of the enzyme. Therefore, it can be expected that the greater the net positive charge in the relevant region of the enzyme, the stronger the interaction of the non-target strand of DNA with the negatively charged phosphodiester skeleton. Amino acids that are not positively charged in the unmodified enzyme can be, for example, amino acids such as neutral, negatively charged, and hydrophobic. Any such amino acid may be replaced with a positively charged amino acid, whereby the desired effect can be achieved. The functional effects described above are based on the complex and related electrostatic and thermodynamic considerations. Therefore, it will be understood that the functional effects described above may be combined. Thus, the CRISPR enzyme can be modified in such a way that it enhances the activity of the enzyme against the target, but also reduces the activity against one or more off-taregts. For example, it is expected that modifications that promote an increase in on-target activity (activity) can be made, while modifications that decrease off-target activity (activity) can be made. Therefore, a synergistic effect can be realized.

It is understood that any of the functional effects described above can be achieved by modifying the amino acids in the groove as described above, but can also be achieved by modifying the amino acids adjacent to or outside the groove. Will be done.

Example 1-Materials and Methods Preparation of SpCas9 Mutants As should be apparent from the overall context of the present disclosure, the abbreviation "SpCas9" refers to Streptococcus pyogenes Cas9, and the abbreviation "SaCas9" is Staphylococcus aureus. Staphylococcus aureus refers to Cas9. Modified SpCas9 and SaCas9 variants, such as modified alanine variants, were created by PCR-based mutagenesis using known techniques (other techniques include Cas9 to express modified or mutated Cas9. The encoding nucleic acid molecule is prepared via a vector expression system, such as, for example, a bacterial expression system or a virus expression vector system, with varying codons for each of one or more protein mutations or one or more modifications. The modified or mutated Cas9 thus expressed can then be immediately purified. The modified or mutated Cas9 of the present invention can be any of the CRISPR-Cas and CRISPR-Cas systems. It can be used in the application of; and advantageously has the advantage that the off-target effect is reduced or virtually absent or essentially absent or absent, and / or the on-target effect is increased. The Cas9 of the present invention or the CRISPR-Cas system of the present invention having the Cas9 of the present invention shall be delivered via a delivery system which may be one or more vectors, including those as discussed herein. Can be done).

Test system for modified Cas9 activity The modified Cas9 enzyme was tested by cotransfecting HEK293T or HEK293FT cells with a plasmid encoding a mutant Cas9 and a plasmid encoding an sgRNA (on-target only). Cells were transfected with Lipofectamine 2000 at 90-95% confluency and harvested by growing at 37 ° C. and 5% CO ₂ for about 72 hours. On-target and off-target genomic loci were PCR amplified and analyzed using next-generation sequencing (NGS). Indel% of on-target and off-target loci were calculated from the sequencing data. SpCas9 mutants were tested at the genomic loci shown in Table A. The SaCas9 mutant is described in Ran at al. Tested by NGS using the EMX101 guide from 2015 and OT1-OT3. No biochemical or SURVEYOR analysis was performed (all data by NGS; Sidi-Chen, "Genome-wise CRISPR Screen in a Mouse Model of Tumor Growth and M. ) ”, Cell 160 (6): 1246-1260, DOI: http: //dx.doi.org/10.10.16/j.cell.2015.02.038,12 March 2015).

Indel analysis Indel analysis by targeted deep sequencing was performed and analyzed as previously described (Hsu, PD et al. (2013), "DNA targeting specificity of DNA targeting specificity of RNA-guided Cas9 nuclease". RNA-guided Cas9 nuclease) ". Nat. Biotechnol. 31,827-832. Cells were harvested approximately 3 days after transfection. Pelleted cells were harvested using the QuickExtract DNA Analysis Kit (Epicentre). QuickExtract (24). Genomic DNA was extracted by resuspending in 80 μL per well, or 20 μL per 96 wells), followed by incubation at 65 ° C. for 15 minutes, 68 ° C. for 15 minutes and 98 ° C. for 10-15 minutes. The PCR fragment of was prepared by a two-step PCR reaction. Briefly, the genomic region of interest was amplified using a primer with a PCR handle for the second round of amplification (Table 2), followed by the fusion PCR method. Added the Illumina P5 adapter and a unique sample-specific bar code to the first round of PCR product.

Example 2-Initial analysis of single SpCas9 mutants (EMX1 and VEGFA target sequences)
Forty-nine first single-point mutations of SpCas9 were made and tested for indel formation. The target sequence was, in this example, the sequences of the EMX1 and VEGFA genes. Table A below shows both the target and off-target sequences along with the PAM sequences. For off-target sequences, the exact mismatches between the target sequences are shown in bold and underlined. The results are shown in FIGS. 2A and 2B.

The following mutants showed reduced activity at off-target sites compared to wild-type enzymes (see FIGS. 2A and 2B).
R63A
H415A
H447A
R778A
R780A
Q807A
K810A
R832A
K848A
K855A
K968A
R976A
H982A
K1000A
K1003A
K1047A
R1060A
K1107A
R1114A
K1118A
K1200A

Example 3-Further analysis of single SpCas9 mutants Several single-point mutation-modified SpCas9 enzymes identified in Example 2 were further analyzed for indel formation at a third guide sequence and additional off-target loci. Tested. The target and off-target sequences are shown in Table A below along with the PAM sequences. For off-target sequences, the exact mismatches between the target sequences are shown in bold and underlined. The results are shown in FIG. The modified SpCas9 enzyme tested in this example was:
R780A
K810A
K848A
K855A
R976A
H982A
K1003A
R1060A

As shown in FIG. 3, all eight modifying enzymes showed reduced activity on off-target sites compared to the unmodified (wild-type) enzyme (SpCas9).

Example 4-Further analysis of single SpCas9 mutant (VEGFA1 target sequence)
Several single-point mutation-modified SpCas9 enzymes, including the mutants described in Example 2, were tested for indel formation at a second different target sequence. In this case, VEGFA1 is the sequence of the VEGFA gene. The modified SpCas9 enzyme tested in this example was:
R780A
K810A
K848A
K855A
R976A
H982A
K1003A
R1060A
H1240A
H1311A

The target and off-target sequences are shown in Table A below along with the PAM sequences. For off-target sequences, the exact mismatches between the target sequences are shown in bold and underlined. The results are shown in FIG.

Example 5-Analysis of Combined SpCas9 Mutants Twenty-four double and 14 triple point mutation-modified SpCas9 enzymes were created and two different target sequences, in this case EMX1 and VEGFAgenes (VEGFA3 is a sequence in VEGFA). Was tested for indel formation in the sequence. The target and off-target sequences are shown in Table A below along with the PAM sequences. For off-target sequences, the exact mismatches between the target sequences are shown in bold and underlined. The mutants tested and the results are shown in FIGS. 4 and 5; the asterisks in FIGS. 4 and 5 indicate embodiments that are currently considered to be advantageous. As shown in FIGS. 4 and 5, all mutants showed a significant reduction in activity against OT 46, OT4, and OT18 off-targets compared to wild-type enzymes. Some combination mutants also showed reduced activity for all four off-targets while maintaining comparable on-target activity as WT; they are:

Example 6-Further analysis of SpCas9 mutants (VEGFA3 target sequence)
Several additional single point mutation-modified SpCas9 enzymes were generated and tested for indel formation on the target sequence VEGFA3, which is the sequence of the VEGFA gene. The modified SpCas9 enzyme tested in this example was as shown below.

As shown in FIG. 2, some mutants showed a significant reduction in activity against both OT1 and OT4 off-targets compared to wild-type enzymes. Some mutants also showed reduced activity against all three off-targets compared to wild-type enzymes, which were:

Analysis of Example 7-SaCas9 mutant (EMX101 target sequence)
Several single point mutant modified SaCas9 enzymes were generated and tested for indel formation on the target sequence, which is the sequence of the EMX101 gene. The target and off-target sequences are shown in Table C below along with the PAM sequences. For off-target sequences, the exact mismatches between the target sequences are shown in bold and underlined. The modified SaCas9 enzyme tested in this example was an alanine mutant as shown below:
K518A
K523A
K525A
H557A
R561A
K572A
R686A
K687A
K692A
R694A
H700A
K751A

As shown in FIG. 6, some mutants showed a significant reduction in activity against both OT2 and OT3 off-targets compared to wild-type enzymes. Some mutants also showed reduced activity against all three off-targets compared to wild-type enzymes, which were:
K523A
K525A
R561A
K572A
R694A
H700A

FIG. 7 provides additional data for the SaCas9 mutant disclosed herein below.

Mutants R245A, R480A, R499A, R650A and R654A showed excellent performance in reducing off-target effects, while R480A, R499A and R245A showed particularly good performance.

Example 8-List of Modified Cas9 Enzymes For SpCas9, the single and combined mutants listed herein have demonstrated favorable specificity improvements, including those in the previous Examples. Therefore, it is considered to be advantageous at present. SpCas9 and SaCas9 mutants, including those tested and others within the present disclosure, are listed in Tables 1-7 below.

Representative examples of SpCas9 mutants are listed in Table 6 below.

Table 7 below provides exemplary mutants in the present disclosure, including those exemplified.

Example 9-Modification of Cas9 for enhanced on-target activity First, we make modifications to uncharged amino acids located in the groove between RuvC-III of SpCas9 and the HNH domain. .. Amino acids that are modified include amino acids with uncharged side chains, including serine, threonine, asparagine and glutamine. Convert the selected amino acid to a positively charged amino acid such as arginine or lysine. The effect of such single amino acid changes on SpCas9 on indel formation at the target locus will be assessed by next-generation sequencing as described above. Select preferred mutations with increased / enhanced on-target activity compared to unmodified enzymes. We evaluate double and triple mutations as described above. Select particularly preferred mutations with enhanced on-target activity.

We evaluate such mutations in SaCas9 in the same manner as described for SpCas9. Select particularly preferred mutations with enhanced on-target activity.

We extend the scope of modification to uncharged amino acids located adjacent to and outside the groove between RuvC-III and the HNH domain of SpCas9. Again, the effect of these changes on indel formation at the target locus is assessed by SURVEYOR analysis as described above. Select preferred mutations with increased / enhanced on-target activity compared to unmodified enzymes. A similar analysis is performed on SaCas9.

We combine the SpCas9 mutation, which indicates enhanced on-target activity, with the mutation, which indicates decreased off-target activity. Again, the effect of these changes on indel formation at the target locus is assessed by SURVEYOR analysis as described above. Select particularly preferred mutations with increased / enhanced on-target activity and reduced off-target activity compared to the unmodified enzyme. A similar analysis is performed on SpCas9.

Analysis of Example 10-SpCas9 mutants (multiple target sequences)
Three mutants, K855A (single mutation), and TM14 and TM15 (both triple mutants) were added to the target sequences EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1. 10, DNMT1.1, DNMT1.2, DNMT1.4, DNMT1.7, VEGFA4, VEGFA5, and VEGFA3 were tested for indel formation. As shown in FIG. 10, all three mutants showed activity against the target and low off-target activity against OT4.

Expanded groups of single, double, and triple mutants were presented with target sequences EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1.10, DNMT1.1, DNMT1.2, DNMT1. Tested for indel formation with .4, DNMT 1.7, VEGFA4, VEGFA5, and VEGFA3. Mutants included E779L, R780A, K810A, K848A, K855A, R976A, H982A, DM11, DM17, DM19, DM20, DM23, DM24, DM25, DM35, DM40, TM14, TM15, and TM16. FIG. 11 summarizes the activity against the target and the off-target activity against OT4. Overall, there was reduced activity for almost all genomic off-targets evaluated, as well as for an exhaustive panel of mismatch guides.

Analysis of Example 11-SpCas9 mutants (VEGFA3 target sequence and off-target sequence)
Several mutant-modified SpCas9 enzymes were generated and tested for indel formation on the target sequence, which is the sequence of the VEGFA3 gene. The modified SpCas9 enzyme tested in this example included:
R780A
K848A
K1000A
K848A R1060A
R780A R1114A
H982A K1003A R1060A
R63A K848A R1060A
R63A K855A R1060A E610G
K1107A
T13I R63A K810A
R63A K855A
R63A H982A
G12D R63A R1060A
H415A K848A
H415A K848A
R780A R1114A
K848A K1107A
K848A E1108A
S1109A
R63A E610G K855A R1060A
R63A K848A R1060A

As shown in FIGS. 12 and 13, some mutants showed a significant reduction in activity against one or more of the three off-target OT1, OT4, and OT18 compared to the wild-type enzyme. .. Some mutants also showed reduced activity against all three off-targets compared to wild-type enzymes. They included:
R780A R1114A
H982A K1003A K1129E
R63A K855A R1060A E610G
K1107A
R63A K855A
R63A H982A
K848A K1107A
R63A E610G K855A R1060A
R63A K848A R1060A

Analysis of Example 12-SpCas9 mutants (EMX1.3 target sequence and off-target sequence)
Several mutant-modified SpCas9 enzymes were tested for indel formation at the target sequence, which is the sequence of EMX1.3. The modified SpCas9 enzyme tested in this example included:
N14K
E779L
E809K
L813R
S845K
L847R
D849A
D861K
E977K
I978K
N979L
N980K

As shown in FIG. 14, certain mutants showed high on-target activity, and in particular, differences in specificity with respect to the off-target sequences OT14, OT23, OT35, OT46, and OT53. Some other mutants demonstrated high activity on off-target sequences.

Analysis of Example 13-SpCas9 mutant (EMX1.3 target)
Several mutant-modified SpCas9 enzymes were tested for indel formation at the target sequence, which is the sequence of the mismatch guide, EMX1.3. The three modified SpCas9 enzymes tested in this example included:
K855A
K810A, K1003A, R1060A
K848A, K1003A, R1060A
The results are shown in FIG.

Example 14-Enhanced Cas9 mutants have high activity and specificity 6 of 29 point mutants compared to wild-type (WT) SpCas9 while maintaining on-target cleavage efficiency. Off-target activity was reduced by at least 10-fold, and the other 6 improved specificity by 2-5-fold. These mutants also exhibited improved specificity when tested at the second locus, VEGFA (1) (Fig. 15D). Some point mutants were more specific than WT SpCas9 when targeting EMX1 (1) and VEGFA (1), but off-target indels were still detectable (about 0.1). %) (Fig. 15D). To further improve specificity, Applicants performed combinatorial mutagenesis using the superior point mutants identified in the initial screening. Eight of the 35 combination mutants retained wild-type on-target activity and exhibited undetectable off-target indel levels in EMX1 (1) OT1, VEGFA (1) OT1, and VEGFA (2) OT2. (Fig. 15E). To confirm that the observed increase in specificity was not due to a decrease in on-target activity, Applicants used the top 16 mutants to generate on-target activity and off-target activity. On-target indel formation at 10 target loci was measured as determined by the combination of (Fig. 15F). Applicants have three mutants: SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (also referred to as eSpCas9 (1.0)), and SpCas9 (K848A / K1003A / R1060A) (eSpCas9 (eSpCas9). High efficiency and specificity were observed for 1.1)). These three variants were selected for further analysis.

To assess whether SpCas9 (K855A), eSpCas9 (1.0), and eSpCas9 (1.1) widely retained efficient nuclease activity, Applicants filed 10 different genomic loci. On-target indel production at 24 target sites across 24 was measured (FIG. 16A). All three mutants produced indel levels comparable to WT SpCas9 in most of the target sites (Fig. 16B). To test whether the improvement in specificity was due to decreased Cas9 expression, Applicants performed Western blots on SpCas9 and all three mutants were equal to or higher than WT SpCas9. It was found that it was expressed at the level (Fig. 16C). This demonstrated that the improvement in specificity was not due to decreased protein expression levels.

Applicants then have specificity for the three mutants in a truncated guide sequence (18 nt for EMX1 (1) and 17 nt for VEGFA (1)) that has been shown to reduce off-target indel formation. Was compared with WT SpCas9. For all three mutants, cleavage was reduced at all off-target sites evaluated. In addition, eSpCas9 (1.0) and eSpCas9 (1.1) removed 20 of 24 of these sites. In contrast, WT SpCas9 with a truncated guide removed 14 of 24 sites, but also increased off-target activity compared to WT SpCas9 with a full-length guide at 5 sites.

To assess tolerance for mismatched target sites of SpCas9 (K855A), eCas9 (1.0), and eCas9 (1.1), Applicants systematically mutated the VEGFA (1) guide sequence. Single and double base mismatches were introduced at various positions (FIGS. 17A-17C). Compared to WT SpCas9, all three mutants induced lower levels of indel in the mismatch guide. Notably, eSpCas9 (1.0) and eSpCas9 (1.1) induced lower indel levels even with a single base mismatch located outside the 7-12 bp seed sequence. Given that Applicants did not observe any difference between eSpCas9 (1.0) and eSpCas9 (1.1) in terms of specificity, SpCas9 (K855A) and based on on-target efficiency. eSpCas9 (1.1) was selected for further analysis.

SpCas9 (K855A) and eSpCas9 (1.1) using BLESS (Direct Insitu Cleavage Label, Enrichment with Streptavidin and Next Generation Sequencing, which Quantifies DNA Double Stranded Cleavage (DSB) Across the Genome) The genome-wide editing specificity of the strain was evaluated (Fig. 17A). Cells were harvested approximately 24 hours after transfection and BLESS was performed. Briefly, a total of 10 million cells were fixed for nuclear isolation and permeabilization, then treated with proteinase K at 37 ° C. for 4 minutes and then inactivated with PMSF. The proteolytic nuclear DSB was labeled overnight with a 200 mM annealed proximal linker. After digesting the labeled nuclei with Proteinase K, chromatin was mechanically sheared with a 26G needle and then sonicated (BioRuptor, 20 minutes high, 50% duty cycle). A total of 20 μg of shear chromatin was captured on streptavidin beads, washed, and ligated to a 200 mM distal linker. Next, the linker hairpin was excised by I-SceI digestion at 37 ° C. for 4 hours, the product was enriched by PCR for 18 cycles, and then the library was prepared with the TruSeq Nano LT kit (Illumina). For negative controls, cells were mock-transfected with Lipofectamine 2000 and pUC19 DNA and processed in parallel throughout the assay.

Calculations of DSB scores to separate background DSBs from authentic Cas9-induced DSBs were performed as previously described (Ran et al, Nature 2015), and loci segregated based on DSB scores, these sgRNAs. The top off-target sites identified earlier for the target have been identified. To provide additional detection capabilities beyond these top off-targets, we have found from previous Cas9-BLESS data that homology search algorithms can help further identify true Cas9-induced DSBs. It was. The homology search algorithm searched for the best matching guide sequence within the region of genome 50 nt on both sides of the median DSB cluster identified by BLESS for all NGG and NAG PAM sequences. Homology-based scores were calculated with the following weights: matches between sgRNA and genomic sequences were scored +3, mismatches were -1, while insertions or omissions between sgRNA and genomic sequences. Loss costs -5. Therefore, an on-target sequence with a complete 20 bp guide + PAM would give a score of 69. The final homology score for the DSB cluster was identified as the maximum score from all possible sequences. Using these weights, we experimentally used a threshold of> 50 for the homology score to create a genuine off-target (indel identified by target deep sequencing) and background. We found that it was completely separated from the DSB. By using this homology criterion for the top 200 BLESS DSB loci, we were able to further identify off-targets from the background DSB.

Applicants assayed EMX1 (1) and VEGFA (1) targets for both mutants and compared their results with WT SpCas9 (FIG. 17B). Both SpCas9 (K855A) and eSpCas9 (1.1) exhibited a genome-wide reduction in off-target cleavage and did not generate any novel off-target sites (FIGS. 17C-17D).

Example 15-Cas9 targeting mechanism and nuclease activity Off-target cleavage occurs when the strength of Cas9 binding to non-target DNA strands exceeds the power of DNA rehybridization. Consistent with this model, mutations designed to weaken the interaction between Cas9 and non-complementary DNA strands lead to a substantial improvement in specificity. The model also suggests that, conversely, the specificity can be reduced by enhancing the interaction between Cas9 and the non-target strand. Consistent with this model, two mutants, S845K and L847R, were created, each exhibiting reduced specificity (FIG. 24).

Example 16-Staphylococcus aureus Specificity of Cas9 (SaCas9) Similar strategies can be applied to other Cas9 family proteins. An improved version of Staphylococcus aureus Cas9 (SaCas9) was created similar to eSpCas9. Single and double amino acid mutants of residues in the groove between the RuvC and HNH domains were screened for reduced off-target cleavage. Mutants with improved specificity were combined to create mutants of SaCas9 that maintained on-target cleavage at EMX site 7 and had significantly reduced off-target cleavage. (FIG. 25) The crystal structure of SaCas9 shows a groove between the HNH domain and the RuvC domain mutated to engineer a nuclease with improved specificity.

Activation of Example 17-HBG1 A complex of spCas9 or spCas9 mutants with various guide RNAs was tested for HBG1 activation. Is FIG. 31 due to a complex containing nuclease-active Cas9 molecules with shortened (ie, "15 bp") guide RNAs (eg, dCas9, R780A / K810A, and R780A / K855A; see also FIG. 4)? , Or activation by a complex of nuclease competent Cas9 (eg, non-mutated spCas9 (px165), R780A, K810A, or K848A). The mutant R780A is noteworthy in that it demonstrated activation in all three guides tested.

Example 18-Particle-mediated delivery of CRISPR-Cas9 components to hematopoietic stem cells (HSCs) Applicants have demonstrated that Cas9 can be delivered to cells by particles. Many nuclear therapeutics may need to deliver both one or more sgRNAs and Cas9 nucleases simultaneously. Therefore, Applicants demonstrate that the complex of modified Cas9 enzyme and sgRNA can be delivered in this manner.

The modified Cas9 enzyme is tested by co-delivering to cells with one or more guide RNAs by particles. The sgRNA targeting the EMX1 gene is mixed with eSpCas9 (1.1) (K848A, K1003A, R1060A) in a 1: 1 molar ratio in sterile nuclease-free 1 × PBS at room temperature for 30 minutes. The control is the same sgRNA mixed with SpCas9. Separately, DOTAP, DMPC, PEG, and cholesterol are dissolved in 100% ethanol. These two solutions are mixed together to form particles containing the Cas9-sgRNA complex. After the particles are formed, HSCs in 96-well plates are transfected with 15 ug of Cas9 protein per well. HSCs are harvested 3 days after transfection, genome-wide editing specificity is evaluated using BLESS, and off-targets are identified by a homology search algorithm. Quantify on-target insertions and deletions (indels) at the EMX1 locus and the number of indels at multiple off-target sites. eSpCas9 (1.1) exhibits a genome-wide reduction in off-target cleavage and no novel off-target sites.

Example 19: Particle-mediated delivery of CRISPR-Cas9 components to hematopoietic stem cells (HSCs) and repair of HBB Two targeting sequences on both sides of a common GAG-> GTG point mutation in the β-globin (HBB) gene The sgRNA is mixed with eSpCas9 (1.1) (K848A, K1003A, R1060A) in a 1: 1 molar ratio of sgRNA vs. enzyme in sterile CRISPR-free 1 × PBS at room temperature for 30 minutes. The control is the same sgRNA mixed with SpCas9. Separately, DOTAP, DMPC, PEG, and cholesterol are dissolved in 100% ethanol. These two solutions and a template nucleic acid for correcting a GAG-> GTG point mutation are mixed together to form a Cas9-sgRNA complex and particles containing the template. After the particles are formed, HSCs in 96-well plates are transfected with 15 ug of Cas9 protein per well. HSCs are harvested 3 days after transfection and tested for repair of GAG-> GTG point mutations. Modified cells are then evaluated for genome-wide editing specificity using BLESS and off-targets are identified by a homology search algorithm. Quantify indels at multiple off-target sites. eSpCas9 (1.1) exhibits a genome-wide reduction in off-target cleavage and no novel off-target sites.

Wild type SpCas9
> K855A
eSpCas9 (1.0)
eSpCas9 (1.1)

Thus, although preferred embodiments of the present invention have been described in detail, the invention as defined by the paragraph above allows many obvious variations thereof without departing from the spirit or scope of the invention. It should be understood that it is not limited to the specific details given in the above description.

Claims (27)

An engineered Cas9 protein containing two or more modifications in the groove between the HNH and the RuvC domain.
Each of the modifications comprises the amino acid substitution of a positively charged residue of wild-type Cas9 with an uncharged residue.
The modification is S.I. Amino acid substitution at a position selected from the group consisting of 780, 810, 848, 855, 976, 982, 1003, and 1060 based on the amino acid position numbering of pyogenes Cas9 (SpCas9).
The engineered Cas9 protein has improved specificity for the target polynucleotide locus as compared to the off-target polynucleotide locus.
Engineered Cas9 protein. The uncharged residue is alanine,
The engineered Cas9 protein according to claim 1. The modification alters the rate of binding reaction at the DNA: RNA: protein interface or alters the energy state of the appropriate RNA: DNA pair.
The engineered Cas9 protein according to claim 1 or 2 . The modifications are based on SpCas9 numbering, R780A and K810A, or R780A and K855A, or R780A and R976A, or K848A and R976A, or K855A and R976A, or R780A and K848A, or K810A and K848A, or K848A. K855A, or K810A and K855A, or H982A and R1060A, or H982A and K1003A , or K1003A and R1060A, or R780A and H982A, or K810A and H982A, or K848A and H982A, or K855A and H982A, or K855A and H982A, or K85A and H982A. Includes amino acid substitutions of K1003A , or K848A and K1003A, or R780A and R1060A, or K810A and R1060A, or K848A and R1060A, or K810A and K1003A.
The engineered Cas9 protein according to any one of claims 1 to 3 . The modifications are based on SpCas9 numbering, H982A, K1003A, and K1129E; or R780A, K1003A, and R1060A; or K810A, K1003A, and R1060A; or K848A, K1003A, and R1060A; or K855A, Includes amino acid substitutions for K1003A and R1060A; or H982A, K1003A, and R1060A.
The engineered Cas9 protein according to any one of claims 1 to 3 . The modification comprises amino acid substitutions of K848A, K1003A, and R1060A, relative to the amino acid positional numbering of SpCas9.
The engineered Cas9 protein according to any one of claims 1 to 3 . The engineered Cas9 proteins are Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Staphylococcus, Parvibaccus, Parvibaccus, Paravibac (Neisseria), Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium from Eubacterium or Corine A mutant of the protein,
The engineered Cas9 protein according to any one of claims 1 to 6 . The engineered Cas9 protein comprises one or more nuclear localization signals (NLS).
The engineered Cas9 protein according to any one of claims 1 to 7 . The engineered Cas9 protein comprises at least two or more NLS.
The engineered Cas9 protein according to claim 8 . The protein comprises three or more modifications.
The engineered Cas9 protein according to any one of claims 1 to 3 . A CRISPR-Cas complex comprising the engineered Cas9 protein according to any one of claims 1 to 10 and a guide polynucleotide . The guide polynucleotide comprises a guide sequence, a tracr mate sequence, and a tracr sequence.
The CRISPR-Cas complex according to claim 11 . The guide polynucleotide is a chimeric guide polynucleotide in which the tracr mate sequence is linked to the tracr sequence.
The CRISPR-Cas complex according to claim 12 . Including making two or more modifications in the groove between the HNH and the RuvC domain.
The method for producing an engineered Cas9 protein according to any one of claims 1 to 10 . A method of reducing off-target activity of the CRISPR-Cas complex.
The method comprises the step of utilizing the engineered Cas9 protein according to any one of claims 1-10 .
The method is not a treatment of the human or animal body,
The method does not include the step of modifying the genetic identity of human germ cell lines.
Method. A method of regulating gene expression in a cell or cell line, or a method of modifying a target sequence.
The step of introducing the CRISPR-Cas complex according to any one of claims 11 to 13 into the cell or cell line is included.
The method is not a treatment of the human or animal body,
The cells are not human germ cell lines
The method does not include the step of modifying the genetic identity of human germ cell lines.
Method. The modified cell or cell line is further cultured to provide a progeny.
16. The method of claim 16 . The cell or cell line is a eukaryotic cell or cell line.
The method according to claim 16 or 17 . The cell or cell line is modified in vivo (ex vivo).
The method according to any one of claims 16 to 18 . A method for producing a plant having a modified trait of interest encoded by a gene of interest.
The CRISPR-Cas complex according to any one of claims 11 to 13 is delivered to a plant cell, whereby the gene of interest is modified, or the modified plant cell is introduced into the cell. The resulting process and
Including the step of regenerating a plant from the plant cells.
Method. Further comprising the step of obtaining seeds from the plant,
The method of claim 20 . A method of modifying a non-human organism by manipulating one or more target sequences.
A step of delivering the CRISPR-Cas complex according to any one of claims 11 to 13 to the organism, thereby obtaining a modified non-human organism.
Here, the method is not a treatment of the animal body,
Method. The organism is a plant or algae,
22. The method of claim 22 . The CRISPR-Cas complex according to any one of claims 11 to 13 for therapeutic use.
The treatment involves gene or genome editing, or gene therapy.
CRISPR-Cas complex. 24. The CRISPR-Cas complex according to claim 24 , wherein the treatment comprises one or more of the following treatments:
Blood or coagulation disorders or disorders; cell dysregulation or oncological disorders or disorders; inflammation or immune-related disorders or disorders; metabolism, liver, kidney or protein disorders or disorders; muscle / skeletal disorders or disorders; neurological or neurological disorders Or disorder; eye disease or disorder. An isolated eukaryote comprising the engineered Cas9 protein according to any one of claims 1-10 or the CRISPR-Cas complex according to any one of claims 11-13 . A cell or eukaryotic cell line. The use of the CRISPR-Cas complex according to any one of claims 11 to 13 for editing a gene or genome.
The use is not a treatment of the human or animal body
The method does not include the step of modifying the genetic identity of human germ cell lines.
use.